Method for transforming stramenopiles

ABSTRACT

Disclosed is a transformation method whereby an ability to produce a useful substance of a stramenopile can be improved. The method for transforming a stramenopile comprises transferring a foreign gene into the stramenopile which is a microorganism belonging to the class  Labyrinthula , more specifically, to a genus  Labyrinthula, Altornia, Aplanochytrium, Schizochytrium, Aurantiochytrium, Thraustochytrium, Ulkenia , etc. Said foreign gene, which is a gene relating to tolerance against an antibiotic, a colorimetric protein and/or a fatty acid desaturase (Δ5 desaturase gene, Δ12 desaturase gene and/or ω3 desaturase gene), is transferred by using the electroporation or gene-gun technique.

TECHNICAL FIELD

The present invention relates to a method for transforming stramenopiles. The invention also relates to stramenopiles having an enhanced unsaturated fatty acid content conferred by the introduction of a fatty acid desaturase gene, and to methods for producing unsaturated fatty acids from such unsaturated fatty acid content-enhanced stramenopiles.

BACKGROUND ART

Polyunsaturated fatty acids (PUFA) represent an important component of animal and human nutrition. ω3 polyunsaturated fatty acids (also called n-3 polyunsaturated fatty acids) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have a wide range of roles in many aspects of health, including brain development in children, eye functions, syntheses of hormones and other signaling substances, and prevention of cardiovascular disease, cancer, and diabetes mellitus (Non-Patent Document 1). These fatty acids therefore represent an important component of human nutrition. Accordingly, there is a need for polyunsaturated fatty acid production.

Meanwhile, microorganisms of the class Labyrinthulomycetes are known to produce polyunsaturated fatty acids. Concerning microorganisms of the family Thraustochytrium, there are reports of, for example, a polyunsaturated fatty acid-containing phospholipid producing process using Schizochytrium microorganisms (Patent Document 1), and Thraustochytrium microorganisms having a docosahexaenoic acid producing ability (Patent Document 2). For enhancement of food and/or feed by the unsaturated fatty acids, there is a strong demand for a simple economical process for producing these unsaturated fatty acids, particularly in the eukaryotic system.

With regard to the class Labyrinthulomycetes, there have been reported foreign gene introducing methods for specific strains of the genus Schizochytrium (the genus Auranthiochytrium (Non-Patent Document 3) in the current classification scheme (Non-Patent Document 2)) (Patent Documents 3 and 4). Further, a method that causes a change in a fatty acid composition by means of transformation is known in which a polyketide synthase (PKS) gene is destroyed to change the resulting fatty acid composition (Non-Patent Document 4). However, there is no report directed to changing a fatty acid composition by manipulating the enzymes of the elongase/desaturase pathway.

CITATION LIST Patent Documents

-   Patent Document 1: JP-A-2007-143479 -   Patent Document 2: JP-A-2005-102680 -   Patent Document 3: JP-A-2006-304685 -   Patent Document 4: JP-A-2006-304686 -   Patent Document 5: JP-A-2005-287380 -   Patent Document 6: PCT/DK96/00051

Non-Patent Documents

-   Non-Patent Document 1: Poulos, A Lipids 30:1-14, 1995; Horrocks, L     A, and Yeo Y K, Pharmacol Res 40:211-225, 1999 -   Non-Patent Document 2: Yokoyama R., Honda D., Mycoscience     48:199-211, 2007 -   Non-Patent Document 3: Lecture Summary for the 60th Conference of     The Society for Biotechnology, Japan, p 136, 2008 -   Non-Patent Document 4: Lippmeier J C et al., Lipids., July;     44(7):621-30. (2009), Epub 2009 Jun. 3. -   Non-Patent Document 5: FEBS Lett. 553, 440-444 (2003). -   Non-Patent Document 6: Nucleic Acids Res. (1994) 22, 4673-4680) -   Non-Patent Document 7: Prasher, D. C. et al., Gene, 111 (2): 229-233     (1992) -   Non-Patent Document 8: Chalfie M. et al., Science, 263:802-805,     (1994) -   Non-Patent Document 9: Southern, P. J., and Berg, P., J. Molec.     Appl. Gen. 1, 327-339. (1982) -   Non-Patent Document 10: Bio-Experiment Illustrated 2, Fundamentals     of Gene Analysis p 63-68, Shujunsha -   Non-Patent Document 11: Sanger, F., et al. Proc. Natl. Acad.     Sci (1977) 74, 5463 -   Non-Patent Document 12: Bio-Experiment Illustrated 2, Fundamentals     of Gene Analysis p 117-128, Shujunsha -   Non-Patent Document 13: Adachi, J. et al. Comput. Sci.     Monogr. (1996) 28

SUMMARY OF THE INVENTION Problems that the Invention is to Solve

The present invention is directed to improving the ability of stramenopiles to produce a useful substance by way of transformation through introduction of a foreign gene. By modifying the ability to produce a useful substance through introduction of a foreign gene associated with the production of a useful substance in stramenopiles, the invention provides a modification method of a fatty acid composition produced by stramenopiles, a method for highly accumulating fatty acids in stramenopiles, an unsaturated fatty acid producing process, stramenopiles having an enhanced unsaturated fatty acid content, and production of unsaturated fatty acid from the unsaturated fatty acid content-enhanced stramenopiles. The present invention provides modification of a fatty acid composition produced by stramenopiles, and a method for highly accumulating fatty acids in stramenopiles, and thus enables more efficient production of polyunsaturated fatty acids.

Means for Solving the Problems

The present inventors conducted intensive studies under the foregoing circumstances of the conventional techniques, and succeeded in transforming stramenopiles with a foreign gene introduced to highly improve the ability to produce an unsaturated fatty acid. The present inventors also found a method for modifying the product fatty acid composition of stramenopiles through expression of a fatty acid desaturase gene introduced into the stramenopiles, and a method for highly accumulating unsaturated fatty acids in the transformed stramenopiles. The present invention was completed after further studies and development for practical applications.

The gist of the present invention includes the following technical matters (1) to (22).

(1) A method for transforming stramenopiles,

comprising introducing a foreign gene into stramenopiles.

(2) The method according to (1), wherein the stramenopiles belong to the class Labyrinthulomycetes.

(3) The method according to (2), wherein the Labyrinthulomycetes are microorganisms belonging to the genus Labyrinthula, Althornia, Aplanochytrium, Japonochytrium, Labyrinthuloides, Schizochytrium, Aurantiochytrium, Thraustochytrium, Ulkenia, Oblongichytrium, Botryochytrium, Parietichytrium, or Sicyoidochytrium.

(4) The method according to anyone of (1) to (3), wherein the microorganisms are any one of a Schizochytrium sp. M-8 strain (FERM P-19755), Thraustochytrium aureum ATCC 34304, Thraustochytrium sp. ATCC 26185, Schizochytrium sp. AL1Ac, Schizochytrium aggregatum ATCC 28209, Ulkenia sp. ATCC 28207, Schizochytrium sp. SEK210 (NBRC 102615), Schizochytrium sp. SEK345 (NBRC 102616), Botryochytrium radiatum SEK353 (NBRC 104107), and Parietichytrium sarkarianum SEK364 (FERM ABP-11298).

(5) The method according to any one of (1) to (4), wherein the foreign gene is a gene associated with tolerance against an antibiotic, colorimetric protein, and/or fatty acid desaturase.

(6) The method according to any one of (1) to (5), wherein the gene associated with fatty acid desaturase is a Δ5 desaturase gene, a Δ12 desaturase gene, and/or an ω3 desaturase gene.

(7) The method according to any one of (1) to (6), wherein the foreign gene is introduced by electroporation or by using a gene gun technique.

(8) A method for modifying the fatty acid composition of stramenopiles,

comprising:

introducing a fatty acid desaturase gene; and

expressing the fatty acid desaturase.

(9) The method according to (8), wherein the fatty acid desaturase is a desaturase.

(10) The method according to (8) or (9), wherein the fatty acid desaturase is a Δ5 desaturase, a Δ12 desaturase, or an ω3 desaturase.

(11) The method according to any one of (8) to (10),

wherein the stramenopiles belong to the class Labyrinthulomycetes.

(12) The method according to (11), wherein the Labyrinthulomycetes are microorganisms belonging to the genus Labyrinthula, Althornia, Aplanochytrium, Japonochytrium, Labyrinthuloides, Schizochytrium, Aurantiochytrium, Thraustochytrium, Ulkenia, Oblongichytrium, Botryochytrium, Parietichytrium, or Sicyoidochytrium.

(13) The method according to (12), wherein the microorganisms are any one of a Schizochytrium sp. M-8 strain (FERM P-19755), Thraustochytrium aureum ATCC 34304, Thraustochytrium sp. ATCC 26185, Schizochytrium sp. AL1Ac, Schizochytrium aggregatum ATCC 28209, Ulkenia sp. ATCC 28207, Schizochytrium sp. SEK210 (NBRC 102615), Schizochytrium sp. SEK345 (NBRC 102616), Botryochytrium radiatum SEK353 (NBRC 104107), and Parietichytrium sarkarianum SEK364 (FERM ABP-11298).

(14) A method for highly accumulating a fatty acid in a stramenopiles by using the method of any one of (8) to (13).

(15) The method according to (14), wherein the fatty acid is an unsaturated fatty acid.

(16) The method according to (15), wherein the unsaturated fatty acid is an unsaturated fatty acid of 18 to 22 carbon atoms.

(17) A fatty acid obtained by using the method of any one of (14) to (16).

(18) Stramenopiles transformed to modify a fatty acid composition.

(19) Stramenopiles transformed to highly accumulate fatty acids.

(20) The stramenopiles according to (18) or (19), wherein the stramenopiles belong to the class Labyrinthulomycetes.

(21) The stramenopiles according to (20), wherein the Labyrinthulomycetes are microorganisms belonging to the genus Labyrinthula, Althornia, Aplanochytrium, Japonochytrium, Labyrinthuloides, Schizochytrium, Aurantiochytrium, Thraustochytrium, Ulkenia, Oblongichytrium, Botryochytrium, Parietichytrium, or Sicyoidochytrium.

(22) The stramenopiles according to (21), wherein the microorganisms are any one of a Schizochytrium sp. M-8 strain (FERM P-19755), Thraustochytrium aureum ATCC 34304, Thraustochytrium sp. ATCC 26185, Schizochytrium sp. AL1Ac, Schizochytrium aggregatum ATCC 28209, Ulkenia sp. ATCC 28207, Schizochytrium sp. SEK210 (NBRC 102615), Schizochytrium sp. SEK345 (NBRC 102616), Botryochytrium radiatum SEK353 (NBRC 104107), and Parietichytrium sarkarianum SEK364 (FERM ABP-11298).

Advantage of the Invention

The present invention enabled modification of the stramenopiles's ability to produce a useful substance (unsaturated fatty acid) through introduction of a foreign gene associated with the production of the useful substance, and thus realized a modification method of a fatty acid composition produced by stramenopiles, and a method for highly accumulating fatty acids in stramenopiles. The invention also realized an unsaturated fatty acid producing process, a stramenopiles having an enhanced unsaturated fatty acid content, and production of an unsaturated fatty acid from the unsaturated fatty acid content-enhanced stramenopiles. The modification of the fatty acid composition produced by stramenopiles, and the method for highly accumulating fatty acid in stramenopiles enabled more efficient production of polyunsaturated fatty acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the results of screening of antibiotics sensitivity test. The labels on the X axis are, from the left, control, G418 (2 mg/ml), Zeocin (1 mg/ml), Puromycin (100 μg/ml), Blasticidin (100 μg/ml), Hygromycin (2 mg/ml), Chloramphnicol (30 μg/ml), Kanamycin (50 μg/ml), Penicillin (500 μg/ml), Streptomycin (500 μg/ml), and Tetracyclin (100 μg/ml).

FIG. 2 represents minimal growth inhibitory concentrations in liquid cultures of T. aureum.

FIG. 3 represents minimal growth inhibitory concentrations in liquid cultures of Thraustochytrium sp.

FIG. 4 represents minimal growth inhibitory concentrations in liquid cultures of mh0186.

FIG. 5 represents minimal growth inhibitory concentrations in liquid cultures of AL1Ac.

FIG. 6 represents minimal growth inhibitory concentrations in plate cultures of T. aureum.

FIG. 7 represents minimal growth inhibitory concentrations in plate cultures of Thraustochytrium sp.

FIG. 8 represents minimal growth inhibitory concentrations in plate cultures of mh0186.

FIG. 9 represents minimal growth inhibitory concentrations in plate cultures of AL1Ac.

FIG. 10 is a schematic view representing a drug-resistant gene cassette (EF-1 α promoter, terminator). Reference numerals: 1. 18S 2. 1R 3. 2F 4. neo-pro-3F 5. n-G-pro-3F 6. n-term-G-4R 7. n-term-G-4F 8. terminator 5R

FIG. 11 is a schematic view representing a drug-resistant gene cassette (ubiquitin promoter, terminator). Reference numerals: 1. Nde118SF 2. 18s-fug-ubq-R 3. Ubpro-HindIII—R 4. UbproG418fus1R 5. ubproG418fus2F 6. G418ubtersus3R 7. G418ubterfus4F 8. KpniterR.

FIG. 12 represents constructed Labyrinthula-Escherichia coli shuttle vectors.

FIG. 13 represents evaluations of A. limacinum transfectants using G418 resistance as an index.

FIG. 14 represents morphological comparisons between A. limacinum transfectants and a wild-type strain.

FIG. 15 represents evaluations of A. limacinum transfectants by PCR using genomic DNA as a template. Reference numerals: 1: Transfectant 1 2: Transfectant 2 3: Transfectant 3 4: Transfectant 4 5: Transfectant 5 6: Wild type 7: Positive control (introduced DNA fragment was used as a template).

FIG. 16 represents evaluations of A. limacinum transfectants by Southern blotting. Reference numerals: (A) 1. Positive control (476-pg introduced DNA) 2. Transfectant 1, Xbaldigestion 3. Transfectant 1, PstI treatment 4. Transfectant 1, HindIII treatment 5. Transfectant 1, EcoRI treatment 6. Transfectant 1, BamHI treatment 7 to 11. negative control (wild type) (B) 1. Positive control (30-pg introduced DNA) 2. Wild type, PstI treatment 3. Transfectant 5, PstI treatment 4. Transfectant 4, PstI treatment 5. Transfectant 3, PstI treatment 6. Transfectant 2, PstI treatment 7. Transfectant 1, PstI treatment.

FIG. 17 represents evaluations of A. limacinum transfectants by RT-PCR. Reference numerals: 1: Transfectant 1 2: Transfectant 2 3: Transfectant 3 4: Transfectant 4 5: Transfectant 5 6: Wild type 7: Positive control (introduced DNA fragment was used as a template); 8 to 13: Total RNA was used as a template.

FIG. 18 represents morphological comparisons between T. aureum transfectants and a wild-type strain.

FIG. 19 represents evaluations of T. aureum transfectants by PCR using genomic DNA as a template, and by Southern blotting. Reference numerals: (A) M: φX174/HincII, λ HindIII 1: No template 2: Positive control (introduced DNA fragment) 3: Transfectant 1 4: Transfectant 2 5: Transfectant 3 6: Wild type (B) P: Positive control (introduced DNA, 2.5 ng) 1: Wild type, NotI treatment 2: Transfectant 1, NotI treatment 3: Transfectant 2, NotI treatment 4: Transfectant 3, NotI treatment.

FIG. 20 represents evaluations of T. aureum transfectants by RT-PCR. Reference numerals: M: φX174/HincII, λ HindIII 1: Transfectant 1 2: Transfectant 2 3: Transfectant 3 4: Wild type 5: Positive control (introduced DNA fragment) 6 to 9: the same as 1 to 4 except that RNA was used as a template in PCR (negative control).

FIG. 21 represents evaluations of Thraustochytrium sp. ATCC 26185 transfectants by PCR using genomic DNA as a template, and by Southern blotting. Reference numerals: (A) 1: λ HindIII digest/φx-174 HincII digest 2: wild type DNA (2F/5R) 3: wild type DNA (only 2F) 4: wild type DNA (only 5R) 5: Transfectant-1 DNA (2F/5R) 6: Transfectant-1 DNA (only 2F) 71: Transfectant-1 DNA (only 5R) 8: Transfectant-2 DNA (2F/5R) 9: Transfectant-2 DNA (only 2F) 10: Transfectant-2 DNA (only 5R) 11: Transfectant-3 DNA (2F/5R) 12: Transfectant-3 RNA (only 2F) 13: Transfectant-3 RNA (only 5R) 14: positive control (2F/5R) 15: positive control (only 2F) 16: positive control (only 5R) (B) 1: λ HindIII digest/φx-174 HincII digest 2: Transfectant-2 DNA (2F/4R) 3: Transfectant-2 DNA (only 2F) 4: Transfectant-2 DNA (only 4R) 5: Transfectant-2 DNA (3F/4R) 6: Transfectant-2 DNA (only 3F) 7: Transfectant-2 DNA (3F/5R) 8: Transfectant-2 DNA (only 5R) (C) 1: wild type, PstI treatment; 2: wild type, HindIII treatment 3: Transfectant-1, PstI treatment 4: Transfectant-1, HindIII treatment 5: Transfectant-2, PstI treatment 6: Transfectant-2, HindIII treatment 71: Transfectant-3, PstI treatment 8: Transfectant-3, HindIII treatment 10: positive control (100-ng introduced DNA).

FIG. 22 represents evaluations of Thraustochytrium sp. ATCC 26185 transfectants by RT-PCR. Reference numerals: (A) 1: λ HindIII digest/φx-174 HincII digest 2: wild type cDNA (3F/4R) 3: wild type cDNA (only 3F) 4: wild type cDNA (only 4R) 5: wild type RNA (3F/4R) 6: wild type RNA (only 3F) 7: wild type RNA (only 4R) 8: Transfectant-1 cDNA (3F/4R) 9: Transfectant-1 cDNA (only 3F) 10: Transfectant-1 cDNA (only 4R) 11: Transfectant-1 RNA (3F/4R) 12: Transfectant-1 RNA (only 3F) 13: Transfectant-1 RNA (only 4R) 14: positive control (3F/4R) 15: positive control (only 3F) 16: positive control (only 4R) (B) 1: ? HindIII digest/φx-174 HincII digest 2: Transfectant-2 cDNA (3F/4R) 3: Transfectant-2 cDNA (only 3F) 4: Transfectant-2 cDNA (only 4R) 5: Transfectant-2 RNA (3F/4R) 6: Transfectant-2 RNA (only 3F) 7: Transfectant-2 RNA (only 4R) 8:Transfectant-3 cDNA (3F/4R) 9:Transfectant-3 cDNA (only 3F) 10: Transfectant-3 cDNA (only 4R) 11: Transfectant-3 RNA (3F/4R) 12: Transfectant-3 RNA (only 3F) 13: Transfectant-3 RNA (only 4R) 14: positive control (3F/4R) 15: positive control (only 3F) 16: positive control (only 4R).

FIG. 23 represents evaluations of Schizochytrium sp. AL1Ac transfectants by PCR using genomic DNA as a template. Reference numerals: Lanes 1 to 3: Transfectant; Lanes 4 to 6: Wild-type strain; Lane 7: No template DNA (negative control); Lane 8: Introduced DNA was used as a template (positive control).

FIG. 24 is a schematic view of a GFP (Green Fluorescent Protein) gene/neomycin-resistant gene expression cassette. Ub-pro-F1 and Ub-term-R2 each include a KpnI site in the sequence.

FIG. 25 represents PCR analyses of a control strain and a GFP gene/neomycin-resistant gene expression cassette-introduced strain, using genomic DNAs derived from these strains as templates. (A, B), PCR results for Aurantiochytrium sp.mh0186; (C, D), PCR results for T. aureum; (A, C), results of amplification of a neomycin-resistant gene; (B, D), results of amplification of a GFP gene. Reference numerals: M: λ HindIII digest/φx-174 HincII digest; N: wild-type strain (negative control); C: neomycin-resistant gene expression cassette-introduced strain (positive control in (A, C); negative control in (B, D)); T: GFP gene/neomycin-resistant gene expression cassette-introduced strain; P: GFP gene/neomycin-resistant gene expression cassette was used as a template (positive control).

FIG. 26 represents PCR analyses of a control strain and a GFP gene/neomycin resistant gene expression cassette-introduced strain, using cDNAs derived from these strains as templates. (A, B), PCR results for Aurantiochytrium sp.mh0186; (C, D), PCR results for T. aureum; (A, C), results of amplification of a neomycin-resistant gene; (B, D), results of amplification of a GFP gene. Reference numerals: M: λ HindIII digest/φx-174 HincII digest; N: wild-type strain (negative control); C: neomycin-resistant gene expression cassette-introduced strain (positive control in (A, C); negative control in (B, D)); T: GFP gene/neomycin-resistant gene expression cassette-introduced strain; P: GFP gene/neomycin-resistant gene expression cassette was used as a template (positive control).

FIG. 27 represents the results of GFP fluorescence observation using a confocal laser microscope. (A), differential interference image of a T. aureum wild-type; (B), fluorescence image of a T. aureum wild-type; (C), differential interference image of GFP expressing T. aureum; (D), fluorescence image of GFP expressing T. aureum; (E), differential interference image of an Aurantiochytrium sp.mh0186 wild-type; (F), fluorescence image of an Aurantiochytrium sp.mh0186 wild-type; (G), differential interference image of GFP expressing Aurantiochytrium sp.mh0186; (H), fluorescence image of GFP expressing Aurantiochytrium sp.mh0186.

FIG. 28 represents multiple alignment analyses for the putative amino acid sequence of Pinguiochrysis pyriformis-derived Δ12 desaturase, and for the amino acid sequences of fungus- and protozoa-derived Δ12 desaturases. Multiple alignment analyses were performed for the amino acid sequences of Δ12 desaturases derived from P. pyriformis, fungus, and protozoan, using ClustalW 1.81 and ESPript 2.2. The same amino acid residues are indicated by blank letters over the solid background, and similar amino acid residues by bold face surrounded by solid lines. Underlines indicate commonly conserved histidine boxes.

FIG. 29 represents phylogenetic analysis of Δ12 desaturase and bifunctional Δ12/Δ15 desaturase.

FIG. 30 represents GC analysis of fatty acid methyl ester (FAME) derived from Saccharomyces cerevisiae to which a control vector pYES2/CT or a recombinant plasmid pYpD12Des was introduced. Arrow indicates a new peak, with a retention time corresponding to that of the sample linoleic acid methyl ester.

FIG. 31 represents GC-MS analysis of a new peak in pYpD12Des-introduced S. cerevisiae-derived FAMEs. Reference numerals: (A), standard substance of linoleic acid; (B), new peak.

FIG. 32 is a schematic view representing a Δ12 desaturase gene/neomycin-resistant gene expression cassette. Ub-pro-F1 and Ub-term-R2 each include a KpnI site in the sequence.

FIG. 33 represents PCR analyses of a control strain and a Δ12 desaturase gene/neomycin-resistant gene expression cassette-introduced strain, using genomic DNAs derived from these strains as templates. (A), results of amplification of neomycin-resistant gene; (B), results of amplification of Δ12 desaturase gene. Reference numerals: M: λ HindIII digest/φx-174 HincII digest; N: wild-type strain (negative control); C1: neomycin-resistant gene expression cassette-introduced strain 1 (positive control in (A); negative control in (B)); C2: neomycin-resistant gene expression cassette-introduced strain 2 (positive control in (A); negative control in (B)); C3: neomycin-resistant gene expression cassette-introduced strain 3 (positive control in (A); negative control in (B)); T1: Δ12 desaturase gene/neomycin-resistant gene expression cassette-introduced strain 1; T2: Δ12 desaturase gene/neomycin-resistant gene expression cassette-introduced strain 2; T3: Δ12 desaturase gene/neomycin-resistant gene expression cassette-introduced strain 3; P: GFP gene/neomycin-resistant gene expression cassette was used as a template (positive control).

FIG. 34 represents PCR analyses of a control strain and a Δ12 desaturase gene/neomycin-resistant gene expression cassette-introduced strain, using cDNAs derived from these strains as templates. (A), results of amplification of neomycin-resistant gene; (B), results of amplification of Δ12 desaturase gene. Reference numerals: M: λ HindIII digest/φx-174 HincII digest; N: wild-type strain (negative control); C1: neomycin-resistant gene expression cassette-introduced strain 1 (positive control in (A); negative control in (B)); C2: neomycin-resistant gene expression cassette-introduced strain 2 (positive control in (A); negative control in (B)); C3: neomycin-resistant gene expression cassette-introduced strain 3 (positive control in (A); negative control in (B)); T1: Δ12 desaturase gene/neomycin-resistant gene expression cassette-introduced strain 1; T2: Δ12 desaturase gene/neomycin-resistant gene expression cassette-introduced strain 2; T3: Δ12 desaturase gene/neomycin-resistant gene expression cassette-introduced strain 3; P: GFP gene/neomycin-resistant gene expression cassette was used as a template (positive control).

FIG. 35 represents multiple alignment of T. aureum-derived Δ5 desaturase.

FIG. 36 represents phylogenetic analysis of desaturase.

FIG. 37 a represents the results of Δ5 desaturase overexpression experiment 1 using yeast as a host. (GC analysis result from ETA-containing medium).

FIG. 37 b represents the results of Δ5 desaturase overexpression experiment 2 using yeast as a host. (GC analysis result using DGLA-containing medium).

FIG. 37 c represents the results of EPA and AA structure analyses by GC-MS; (a), TauΔ5des product EPA; (b), EPA standard substance: (c), TauΔ5des product AA; (d), AA standard substance.

FIG. 38, (a), represents a vector construct containing a Δ5 desaturase gene/neomycin-resistant gene expression cassette; (b), a PCR amplified Δ5 desaturase gene/neomycin-resistant gene expression cassette.

FIG. 39 represents PCR analyses of a control strain and a Δ5 desaturase gene/neomycin-resistant gene expression cassette-introduced strain, using genomic DNAs derived from these strains as templates. Lanes 1 to 6, amplified neomycin-resistant gene; Lanes 7 to 12, amplified Δ5 desaturase gene. Reference numerals: 1: Δ5 desaturase gene/neomycin-resistant gene expression cassette-introduced strain 1; 2: Δ5 desaturase gene/neomycin-resistant gene expression cassette-introduced strain 2; 3: Δ5 desaturase gene/neomycin-resistant gene expression cassette-introduced strain 3; 4: wild-type strain (negative control); 5: Δ5 desaturase gene/neomycin-resistant gene expression cassette was used as a template (positive control); 6: No template (negative control); 7: Δ5 desaturase gene/neomycin-resistant gene expression cassette-introduced strain 1; 8: Δ5 desaturase gene/neomycin-resistant gene expression cassette-introduced strain 2; 9: Δ5 desaturase gene/neomycin-resistant gene expression cassette-introduced strain 3; 10: wild-type strain (negative control); 11: Δ5 desaturase gene/neomycin-resistant gene expression cassette was used as a template (positive control); 12: No template (negative control).

FIG. 40 represents PCR analyses of a control strain and a Δ5 desaturase gene/neomycin-resistant gene expression cassette-introduced strain, using cDNAs derived from these strains as templates. The upper panel represents the results of amplification of neomycin-resistant gene, and the lower panel represents the results of amplification of Δ5 desaturase gene. Reference numerals: mhneo^(r)1: neomycin-resistant gene expression cassette-introduced strain 1; mhneo^(r)2: neomycin-resistant gene expression cassette-introduced strain 2; mhneo^(r)3: neomycin-resistant gene expression cassette-introduced strain 3; mhΔ5neo^(r)1: Δ5 desaturase gene/neomycin-resistant gene expression cassette-introduced strain 1; mhΔ5neo^(r)2: Δ5 desaturase gene/neomycin-resistant gene expression cassette-introduced strain 2; mhΔ5neo^(r)3: Δ5 desaturase gene/neomycin-resistant gene expression cassette-introduced strain 3.

FIG. 41 represents GC analyses of FAMEs derived from a control strain or a Δ12 desaturase gene/neomycin-resistant gene expression cassette-introduced Aurantiochytrium sp.mh0186. Arrow indicates a new peak, with a retention time corresponding to that of the sample linoleic acid methyl ester.

FIG. 42 represents GC-MS analyses of a new peak in Δ12 desaturase gene/neomycin-resistant gene expression cassette-introduced strain-derived FAMEs.

FIG. 43 compares fatty acid compositions of a control strain and a Δ12 desaturase gene/neomycin-resistant gene expression cassette-introduced strain. The blank bar and solid bar represent the fatty acid compositions of the control strain and the Δ12 desaturase gene/neomycin-resistant gene expression cassette-introduced strain, respectively. Arrow indicates the foreign fatty acid oleic acid, and the star the biosynthesized linoleic acid. Values are given as mean values±standard deviation.

FIG. 44 represents the results of the GC analysis of a mh0186 transfectant.

FIG. 45 represents the results of Neo^(r) (about 2,300 bp) detection by PCR, showing that specific Neo^(r) amplification, not found in the wild-type strain, was observed in the gene-introduced Labyrinthula transfectants.

FIG. 46 represents a plasmid containing an SV40 terminator sequence derived from a subcloned pcDNA 3.1 Myc-His vector.

FIG. 47 is a schematic view representing primers used for Fusion PCR, and the product. The end product had a fused sequence of Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter and pTracer-CMV/Bsd/lacZ-derived blasticidin resistant gene.

FIG. 48 represents a pTracer-CMV/Bsd/lacZ-derived blasticidin resistant gene BglII cassette produced.

FIG. 49 is a schematic view representing primers used for Fusion PCR, and the product. The end product had a fused sequence of Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter, Saprolegnia diclina-derived ω3 desaturase gene sequence, and Thraustochytrium aureum ATCC 34304-derived ubiquitin terminator.

FIG. 50 represents a plasmid in which one of the BglII sites in the blasticidin resistant gene BglII cassette of FIG. 48 is replaced with a KpnI site.

FIG. 51 represents a Saprolegnia diclina-derived ω3 desaturase expression plasmid produced. The plasmid includes a blasticidin resistant gene as a drug resistance marker.

FIG. 52 is a schematic view representing positions of the primers used for a PCR performed to confirm insertion of Saprolegnia diclina-derived ω3 desaturase gene into the genome.

FIG. 53 represents evaluations of a Thraustochytrium aureum ATCC 34304 transfectant strain by PCR using genomic DNA as a template. Reference numerals: Lanes 1 and 2: transfectant.

FIG. 54 compares the fatty acid compositions of a Thraustochytrium aureum ATCC 34304 control strain and an (03 desaturase gene introduced strain. The blank bar and solid bar represent the fatty acid compositions of the control strain and the ω3 desaturase gene introduced strain, respectively. Values are given as mean values±standard deviation.

FIG. 55 represents the percentage of fatty acids in the control strain and the ω3 desaturase gene introduced strain relative to the percentage of the Thraustochytrium aureum ATCC 34304 wild-type strain taken as 100%.

MODE FOR CARRYING OUT THE INVENTION

The recent studies of the physiological activity and the pharmacological effects of lipids have elucidated the conversion of unsaturated fatty acids into various chemical substances, and the roles of unsaturated fatty acids in the unsaturated fatty acid metabolism. Particularly considered important in relation to disease is the nutritionally preferred proportions of saturated fatty acids, monounsaturated fatty acids, and unsaturated fatty acids, and the proportions of fish oil-derived ω3 series (also known as the n-3 series) fatty acids such as eicosapentaenoic acid and docosahexaenoic acid, and plant-derived ω6 series (also known as the n-6 series) fatty acids as represented by linoleic acid. Because animals are deficient in fatty acid desaturases (desaturases) or have low levels of fatty acid desaturases, some unsaturated fatty acids need to be ingested with food. Such fatty acids are called essential fatty acids (or vitamin F), which include linoleic acid (LA), γ-linolenic acid (GLA), and arachidonic acid (AA or ARA).

Unsaturated fatty acid production involves enzymes called fatty acid desaturases (desaturases). The fatty acid desaturases (desaturases) are classified into two types: (1) those creating a double bond (also called an unsaturated bond) at a fixed position from the carbonyl group of a fatty acid (for example, Δ9 desaturase creates a double bond at the 9th position as counted from the carbonyl side), and (2) those creating a double bond at a specific position from the methyl end of a fatty acid (for example, ω3 desaturase creates a double bond at the 3rd position as counted from the methyl end). It is known that the biosynthesis of unsaturated fatty acid involves the repetition of a set of two reactions, the creation of a double bond by the desaturase (unsaturation), and the elongation of the chain length by several different elongases. For example, Δ9 desaturase synthesizes oleic acid (OA) by unsaturating the stearic acid either synthesized in the body from palmitic acid or ingested directly. Δ6, Δ5, and Δ4 desaturases are fatty acid desaturases (desaturases) essential for the syntheses of polyunsaturated fatty acids such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).

The Labyrinthulomycetes, a member of stramenopiles, has two families: Thraustochytrium (Thraustochytriaceae) and Labyrinthulaceae. These microorganisms are known to accumulate polyunsaturated fatty acids such as arachidonic acid, EPA, DTA, DPA, and DHA.

The present invention is concerned with a stramenopiles transformation method that introduces a foreign gene into a stramenopiles. The transformation method of the present invention is the basis for providing a novel modification method of a fatty acid composition produced by stramenopiles, a novel method for highly accumulating fatty acids in a stramenopiles, and a novel unsaturated fatty acid producing process. The transformation method has also made it possible to develop and provide a stramenopiles having an enhanced unsaturated fatty acid content conferred by the introduction of a fatty acid desaturase gene, and a method for producing unsaturated fatty acids from the unsaturated fatty acid content-enhanced stramenopiles.

The present invention is described below in more detail.

[Microorganism]

The microorganisms used in the fatty acid modification method of the present invention are not particularly limited, as long as the microorganisms are stramenopiles considered to carry out fatty acid synthesis after introduction of a fatty acid desaturase gene. Particularly preferred microorganisms are those belonging to the class Labyrinthulomycetes. Examples of the Labyrinthulomycetes include those of the genus Labyrinthula, Althornia, Aplanochytrium, Japonochytrium, Labyrinthuloides, Schizochytrium, Thraustochytrium, Ulkenia, Aurantiochytrium, Oblongichytrium, Botryochytrium, Parietichytrium, and Sicyoidochytrium.

The stramenopiles used in the present invention are preferably those belonging to the genus Schizochytorium, Thraustochytrium, Aurantiochytrium, and Parietichytrium, particularly preferably a Schizochytrium sp. M-8 strain (FERM P-19755), Thraustochytrium aureum ATCC34304, Thraustochytrium sp. ATCC26185, Schizochytrium sp. AL1Ac, Schizochytrium aggregatum ATCC28209, Ulkenia sp. ATCC 28207, Schizochytrium sp. SEK210 (NBRC 102615), Schizochytrium sp. SEK345 (NBRC 102616), Botryochytrium radiatum SEK353 (NBRC 104107), and Parietichytrium sarkarianum SEK364 (FERM ABP-11298). The Schizochytrium sp. M-8 strain is reported in Patent Document 5, and was acquired according to the method described in this publication (Thraustochytrium M-8 strain). First, the sea water and fallen leaves collected in the mangrove forest on Ishigakijima were placed in a 300-ml Erlenmeyer flask, and about 0.05 g of pine pollens (collected at the shore near the city of Miyazaki) were added. The sample was left unattended at room temperature for one week, and the sea water was collected with the pine pollens floating on the surface. The water (0.1 ml) was then applied onto a potato dextrose agar medium prepared in a petri dish. The sample was cultured at 28° C. for 5 days, and cream-colored, non-glossy colonies were picked up, and applied onto a new agar medium. After 3 days, the proliferated microorganisms were observed under a microscope, and preserved in a slant medium after determining the microorganisms as Labyrinthulomycetes from the cell size and morphology. Note that this strain has been domestically deposited, and is available from The National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (Tsukuba Center, Chuou Dairoku, 1-1-1, Higashi, Tsukuba-shi, Ibaraki) (accession number: FERM P-19755; Mar. 29, 2004). The Parietichytrium sarkarianum SEK364 strain was obtained from the surface water collected at the mouth of fukidougawa on Ishigakijima. The water (10 ml) was placed in a test tube, and left unattended at room temperature after adding pine pollens. After 7 days, the pine pollens were applied to a sterile agar medium (2 g glucose, 1 g peptone, 0.5 g yeast extract, 0.2 g chloramphenicol, 15 g agar, distilled water 100 mL, sea water 900 mL). Colonies appearing after 5 days were isolated, and cultured again. This was repeated several times to isolate the cells. Note that this strain has been internationally deposited, and is available from The National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (Tsukuba Center, Chuou Dairoku, 1-1-1, Higashi, Tsukuba-shi, Ibaraki) (accession number: FERM ABP-11298; Sep. 24, 2010).

It should be noted that the stramenopiles are also referred to by other names in literatures: Schizochytorium sp. mh0186, Aurantiochytirum sp. mh0186, or Aurantiochytrium limacinum mh0186. These names are also referred to in the present invention. These stramenopiles are cultured in common media, including solid medium and liquid medium, using an ordinary method. The type of medium used is not particularly limited, as long as it is one commonly used for culturing Labyrinthulomycetes, and that contains, for example, a carbon source (such as glucose, fructose, saccharose, starch, and glycerine), a nitrogen source (such as a yeast extract, a corn steep liquor, polypeptone, sodium glutamate, urea, ammonium acetate, ammonium sulfate, ammonium nitrate, ammonium chloride, and sodium nitrate), an inorganic salt (such as potassium phosphate) and appropriately combined with other necessary components. The prepared medium is adjusted to a pH of 3.0 to 8.0, and used after being sterilized with an autoclave or the like. The Thraustochytrium aureum ATCC 34304, Thraustochytrium sp. ATCC 26185, Schizochytrium aggregatum ATCC 28209, and Ulkenia sp. ATCC 28207 are deposited and available from ATCC. The Schizochytrium sp. SEK210 (NBRC 102615), Schizochytrium sp. SEK345 (NBRC 102616), and Botryochytrium radiatum SEK353 (NBRC 104107) are deposited and available from The National Institute of Technology and Evaluation.

[Fatty Acid Desaturase]

The fatty acid desaturase (desaturase) of the present invention is not particularly limited, as long as it functions as a fatty acid desaturase. The origin of the fatty acid desaturase gene is not particularly limited, and may be, for example, animals and plants. Examples of the preferred fatty acid desaturase genes include Δ4 fatty acid desaturase gene, Δ5 fatty acid desaturase gene, Δ6 fatty acid desaturase gene, and Δ12 fatty acid desaturase gene, and these may be used either alone or in combination. The Δ4 fatty acid desaturase gene, Δ5 fatty acid desaturase gene, Δ6 fatty acid desaturase gene, and Δ12 fatty acid desaturase gene create an unsaturated bond at carbon 4, 5, 6, and 12, respectively, as counted from the terminal carboxyl group (delta end) of the fatty acid. A specific example of these fatty acid desaturase genes is the microalgae-derived Δ4 fatty acid desaturase gene (Tonon, T., Harvey, D., Larson, T. R., and Graham, I. A. Identification of a very long chain polyunsaturated fatty acid Δ4-desaturase from the microalga Pavlova lutheri; Non-Patent Document 5). Specific examples of Δ5 desaturase include T. aureum-derived desaturase, and Δ5 desaturases derived from Thraustochytrium sp. ATCC 26185, Dictyostelium discoideum, Rattus norvegicus, Mus musculus, Homo sapiens, Caenorhabditis elegans, and Leishmania major. Examples of Δ12 desaturase include Pinguiochrysis pyriformis-derived Δ12 desaturase, and fungus- and protozoa-derived Δ12 desaturases.

Desaturase is essential for the production of polyunsaturated fatty acids having many important functions. For example, polyunsaturated fatty acids are the main component of the cell membrane, and exist in the form of phospholipids. The fatty acids also function as precursor substances of mammal prostacyclin, eicosanoid, leukotriene, and prostaglandin. Polyunsaturated fatty acids are also necessary for the proper development of a growing infant brain, and tissue formation and repair. Given the biological significance of the polyunsaturated fatty acids, there have been attempts to efficiently produce polyunsaturated fatty acids, and intermediates of polyunsaturated fatty acids.

Δ5 desaturase catalyzes, for example, the conversion of dihomo-γ-linolenic acid (DGLA) to arachidonic acid (AA), and the conversion of eicosatetraenoic acid (ETA) to eicosapentaenoic acid (EPA). Δ6 desaturase catalyzes, for example, the conversion of linoleic acid (LA) to γ-linolenic acid (GLA), and the conversion of α-linolenic acid (ALA) to stearidonic acid (STA). Aside from Δ5 desaturase and Δ6 desaturase, many other enzymes are involved in the polyunsaturated fatty acid biosynthesis. For example, elongase catalyzes the conversion of γ-linolenic acid (GLA) to dihomo-γ-linolenic acid (DGLA), and the conversion of stearidonic acid (STA) to eicosatetraenoic acid (ETA). Linoleic acid (LA) is produced from oleic acid (OA) by the action of Δ12 desaturase.

[Product Unsaturated Fatty Acid]

The unsaturated fatty acid produced by the fatty acid desaturase expressed in stramenopiles are, for example, an unsaturated fatty acid of 18 to 22 carbon atoms. Preferred examples include docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), though the preferred unsaturated fatty acids vary depending on the types of the fatty acid desaturase and the fatty acid substrate used. Other examples include α-linolenic acid (ALA), octadecatetraenoic acid (OTA, 18:4n-3), eicosatetraenoic acid (ETA, 20:4n-3), n-3 docosapentaenoic acid (DPA, 22:5n-3), tetracosapentaenoic acid (TPA, 24:5n-3), tetracosahexaenoic acid (THA, 24:6n-3), linoleic acid (LA), γ-linolenic acid (GLA), eicosatrienoic acid (20:3n-6), arachidonic acid (AA), and n-6 docosapentaenoic acid (DPA, 22:5n-6).

[Fatty Acid Desaturase Gene Source]

The organisms that can be used as the fatty acid desaturase gene source in the present invention are not limited to particular genus, species, or strains as described in paragraph [0021], and may be any organisms having an ability to produce polyunsaturated fatty acids. For example, in the case of microorganisms, such organisms are readily available from microorganism depositary authorities. Examples of such microorganisms include the bacteria Moritella marina MP-1 strain (ATCC15381) of the genus Moritella. The following describes a method using this strain as an example of desaturase and elongase gene sources. The method, however, is also applicable to the isolation of the constituent desaturase and elongase genes from all biological species having the desaturase/elongase pathway.

Isolation of the desaturase and/or elongase gene from the MP-1 strain requires estimation of a conserved region in the amino acid sequence of the target enzyme gene. For example, in desaturase, it is known that a single cytochrome b5 domain and three histidine boxes are conserved across biological species, and that elongase has two conserved histidine boxes across biological species. More specifically, the conserved region of the target enzyme can be estimated by the multiple alignment comparison of the known amino acid sequences of the desaturase or elongase genes derived from various biological species using the clustal w program (Thompson, J. D., et al.; Non-Patent Document 6). It is also possible to estimate conserved regions specific to desaturase and/or elongase having the same substrate specificity by the multiple alignment comparison of the amino acid sequences of desaturase or elongase genes having the same substrate specificity in the desaturase and/or elongase derived from known other organisms. Various degenerate oligonucleotide primers are then produced based on the estimated conserved regions, and the partial sequence of the target gene derived from the MP-1 strain is amplified using an MP-1 strain-derived cDNA library as a template, by using methods such as PCR and RACE. The resulting amplification product is cloned into a plasmid vector, and the base sequence is determined using an ordinary method. The sequence is then compared with a known enzyme gene to confirm isolation of a part of the target enzyme gene from the MP-1 strain. The full-length target enzyme gene can be obtained by hybridization screening using the obtained partial sequence as a probe, or by the RACE technique using the oligonucleotide primers produced from the partial sequence of the target gene.

[Other Gene Sources]

Reference should be made to Non-Patent Document 7 or 8 for GFP (Green Fluorescent Protein), Patent Document 6 for EGFP (enhanced GFP), and Non-Patent Document 9 for neomycin-resistant gene.

[Introduction and Expression of Fatty Acid Desaturase in Stramenopiles]

The fatty acid desaturase gene may be introduced by way of transformation using the conventional method of gene introduction into a microorganism. An example of such a method is the transformation introducing a recombinant expression vector into a cell. Details of the desaturase gene introduction into stramenopiles in the present invention will be specifically described later in Examples. The stramenopiles used for transformation are not particularly limited, and those belonging to the class Labyrinthulomycetes can preferably be used, as described above.

The expression vector is not particularly limited, and a recombinant expression vector with an inserted gene may be used. The vehicle used to produce the recombinant expression vector is not particularly limited, and, for example, a plasmid, a phage, and a cosmid may be used. A known method may be used for the production of the recombinant expression vector. The vector is not limited to specific types, and may be appropriately selected from vectors expressible in a host cell. Specifically, the expression vector may be one that is produced by incorporating the gene of the present invention into a plasmid or other vehicles with a promoter sequence appropriately selected according to the type of the host cell for reliable expression of the gene. The expression vector preferably includes at least one selection marker. Examples of the marker for eukaryotic cell cultures include dihydrofolate reductase, a neomycin-resistant gene, and a GFP. In consideration of the results for antibiotic sensitivity and the selection marker genes used in the eukaryotes transformation system, the selection marker genes presented in Table 1 below were shown to be effective for the Labyrinthulomycetes transformation system.

These selection markers allow for confirmation of whether the polynucleotide according to the present invention has been introduced into a host cell, or whether the polynucleotide is reliably expressed in the host cell. Alternatively, the fatty acid desaturase according to the present invention may be expressed as a fused polypeptide. For example, the fatty acid desaturase according to the present invention may be expressed as a GFP fused polypeptide, using an Aequorea-derived green fluorescence polypeptide GFP as a marker.

Preferably, the foreign gene is introduced by electroporation or by using the gene gun technique. Specific introduction conditions are presented in Table 2. In the present invention, the introduction of the fatty acid desaturase gene changes the fatty acid composition of the cell from that before the introduction of the fatty acid desaturase gene. Specifically, the fatty acid composition is modified by the expression of the fatty acid desaturase gene.

The stramenopiles transformation produce a stramenopiles (microorganism) in which the composition of the fatty acid it produces is modified. The stramenopiles with the fatty acid desaturase-encoding gene expressibly introduced therein can be used for, for example, the production of unsaturated fatty acids. Unsaturated fatty acid production is possible with the stramenopiles that has been modified to change its fatty acid composition as above, and other conditions, including manufacturing process, equipment, and instruments are not particularly limited. The unsaturated fatty acid production includes the step of culturing a microorganism that has been modified to change its fatty acid composition by the foregoing modification method, and the microorganism is used with a medium to produce unsaturated fatty acids.

The cell culture conditions (including medium, culture temperature, and aeration conditions) may be appropriately set according to such factors as the type of the cell, and the type and amount of the unsaturated fatty acid to be produced.

As used herein, the term “unsaturated fatty acids” encompasses substances containing unsaturated fatty acids, and attributes such as the content, purity, shape, and composition are not particularly limited. Specifically, in the present invention, the cell or medium itself having a modified fatty acid composition may be regarded as unsaturated fatty acids. Further, a step of purifying the unsaturated fatty acids from such cells or media also may be included. A known method of purifying unsaturated fatty acids and other lipids (including conjugate lipids) may be used for the purification of the unsaturated fatty acids.

[Method of Highly Accumulating Unsaturated Fatty Acid in Stramenopiles]

Accumulation of unsaturated fatty acids in stramenopiles are realized by culturing the transformed stramenopiles of the present invention. For example, the culture is performed using a common solid or liquid medium. The type of medium used is not particularly limited, as long as it is one commonly used for culturing Labyrinthulomycetes, and that contains, for example, a carbon source (such as glucose, fructose, saccharose, starch, and glycerine), a nitrogen source (such as a yeast extract, a corn steep liquor, polypeptone, sodium glutamate, urea, ammonium acetate, ammonium sulfate, ammonium nitrate, ammonium chloride, and sodium nitrate), an inorganic salt (such as potassium phosphate), and appropriately combined with other necessary components. Particularly preferably, a yeast extract/glucose agar medium (GY medium) is used. The prepared medium is adjusted to a pH of 3.0 to 8.0, and used after being sterilized with an autoclave or the like. The culture may be performed by aerated stirred culture, shake culture, or static culture at 10 to 40° C., preferably 15 to 35° C., for 1 to 14 days.

For the collection of the produced unsaturated fatty acids, the stramenopiles are grown in a medium, and the intracellular lipids (oil and fat contents with the polyunsaturated fatty acids, or the polyunsaturated fatty acids) are released by processing the microorganism cells obtained from the medium. The lipids are then collected from the medium containing the released intracellular lipids. Specifically, the cultured stramenopiles are collected by using a method such as centrifugation. The cells are then disrupted, and the intracellular fatty acids are extracted using a suitable organic solvent according to an ordinary method. Oil and fat with the enhanced polyunsaturated fatty acid content can be obtained in this manner.

In the present invention, the transformed stramenopiles with the introduced fatty acid desaturase gene are cultured, and the stramenopiles produce fatty acids of a modified composition. This is the result of the introduced fatty acid desaturase unsaturating the fatty acids normally produced in stramenopiles. The fatty acid compositional changes before and after the modification are presented and compared in Tables to 10. For example, although the expression of Pinguiochrysis-derived Δ12 desaturase does not change the types of the fatty acids produced, the introduced enzyme affects the product ratio. Specifically, oleic acid was converted to linoleic acid, at a conversion efficiency of 30±6.6%.

In an expression test using a foreign Labyrinthula-derived Δ5 desaturase in a particular species of Labyrinthula, the EPA content showed an about 1.4-fold increase. In a culture performed in a medium containing ETA or DGLA, the ETA and DGLA were converted to EPA and AA, respectively, and the unsaturated fatty acids increased. As to the conversion efficiency in Labyrinthula, the conversion efficiency of a precursor substance in Labyrinthula was higher than that in a yeast, specifically 75% for ETA, and 63% for DGLA. These results were obtained form CG-MS test data.

The unsaturated fatty acids of the present invention encompass various drugs, foods, and industrial products, and the applicable areas of the unsaturated fatty acids are not particularly limited. Examples of the food containing oil and fat that contain the unsaturated fatty acids of the present invention include foods with health claims such as supplements, and food additives. Examples of the industrial products include feeds for non-human organisms, films, biodegradable plastics, functional fibers, lubricants, and detergents.

The present invention is described below in more detail based on examples. Note, however, that the present invention is in no way limited by the following examples.

Example 1 Labyrinthulomycetes, Culture Method, and Preservation Method (1) Strains Used in the Present Invention

Thraustochytrium aureum ATCC 34304, and Thraustochytrium sp. ATCC 26185 were obtained from ATCC. Aurantiochytrium limacinum mh0186, and Schizochytrium sp. AL1Ac were obtained from University of Miyazaki, Faculty of Agriculture.

Schizochytrium aggregatum ATCC 28209, and Ulkenia sp. ATCC 28207 were obtained from ATCC. Schizochytrium sp. SEK210 (NBRC 102615), Schizochytrium sp. SEK345 (NBRC 102616), Botryochytrium radiatum SEK353 (NBRC 104107), and Parietichytrium sarkarianum SEK364 were obtained from Konan University, Faculty of Science and Engineering.

(2) Medium Composition

i. Agar Plate Medium Composition

PDA Agar Plate Medium

A 0.78% (w/v) potato dextrose agar medium (Nissui Pharmaceutical Co., Ltd.), 1.75% (w/v) Sea Life (Marine Tech), and a 1.21% (w/v) agar powder (nacalai tesque) were mixed, and sterilized with an autoclave at 121° C. for 20 min. After sufficient cooling, ampicillin sodium (nacalai tesque) was added in a final concentration of 100 μg/ml to prevent bacterial contamination. The medium was dispensed onto a petri dish, and allowed to stand on a flat surface to solidify.

ii. Liquid Medium Composition

GY Liquid Medium

3.18% (w/v) glucose (nacalai tesque), a 1.06% (w/v) dry yeast extract (nacalai tesque), and 1.75% (w/v) Sea Life (Marine Tech) were mixed, and sterilized with an autoclave at 121° C. for 20 min. Then, 100 μg/ml ampicillin sodium (nacalai tesque) was added.

PD Liquid Medium

0.48% (w/v) potato dextrose (Difco), and 1.75% (w/v) Sea Life (Marine Tech) were mixed, and sterilized with an autoclave at 121° C. for 20 min. Then, 100 μg/ml ampicillin sodium (nacalai tesque) was added.

H Liquid Medium

0.2% (w/v) glucose (nacalai tesque), a 0.02% (w/v) dry yeast extract (nacalai tesque), 0.05% sodium glutamate (nacalai tesque), and 1.75% (w/v) Sea Life (Marine Tech) were mixed, and sterilized with an autoclave at 121° C. for 20 min. Then, 100 μg/ml ampicillin sodium (nacalai tesque) was added.

(3) Culture Method

i. Agar Plate Culture

Labyrinthula cells were inoculated using a platinum loop or a spreader, and static culture was performed at 25° C. to produce colonies. Subcultures were produced by collecting the colonies with a platinum loop, suspending the collected colonies in a sterilized physiological saline, and applying the suspension using a platinum loop or a spreader. As required, the cells on the plate were inoculated in a liquid medium for conversion into a liquid culture.

ii. Liquid Culture

Labyrinthula cells were inoculated, and suspension culture was performed by stirring at 25° C., 150 rpm in an Erlenmeyer flask or in a test tube. Subcultures were produced by adding a culture fluid to a new GY or PD liquid medium in a 1/200 to 1/10 volume after confirming proliferation from the logarithmic growth phase to the stationary phase. As required, the cell culture fluid was applied onto a PDA agar plate medium for conversion into an agar plate culture.

(4) Maintenance and Preservation Method of Labyrinthulomycetes

In addition to the subculture, cryopreservation was performed by producing a glycerol stock. Specifically, glycerol (nacalai tesque) was added in a final concentration of 15% (v/v) to the logarithmic growth phase to stationary phase of a cell suspension in a GY liquid medium, and the cells were preserved in a −80° C. deep freezer.

Example 2 Selection of Selection Markers Used for Antibiotic Sensitivity Test and for Transformation System of Labyrinthulomycetes (1) Screening of Antibiotics Showing Sensitivity in Liquid Culture

Precultures of four strains of Labyrinthulomycetes were added to GY liquid media containing various antibiotics, and cultured at 150 rpm, 25° C. for 5 days. Then, turbidity at 600 nm (OD600) was measured. FIG. 1 presents the antibiotics used and antibiotic concentrations, along with the measurement results.

(2) Determination of Minimal Growth Inhibitory Concentration (MIC) in Liquid Culture

MICS in liquid culture were determined for the antibiotics that Labyrinthulomycetes showed sensitivity. Precultures of four strains of Labyrinthulomycetes were added to GY liquid media containing various antibiotics of different concentrations, and cultured at 150 rpm, 25° C. for 5 days. Then, turbidity at 600 nm (OD₆₀₀) was measured. FIG. 2 present the results for T. aureum, FIG. 3 present the results for Thraustochytrium sp. ATCC 26185, FIG. 4 present the results for A. limacinum mh0186, and FIG. 5 present the results for Schizochytrium sp. AL1Ac, respectively.

(3) Determination of MIC in Agar Plate Culture

Precultures (5 μl) of four strains of Labyrinthulomycetes were dropped onto PDA agar media containing various antibiotics of different concentrations, and observed for colony formation after being cultured at 25° C. for 7 days. FIG. 6 present the results for T. aureum, FIG. 7 present the results for Thraustochytrium sp. ATCC 26185, FIG. 8 present the results for A. limacinum mh0186, and FIG. 9 present the results for Schizochytrium sp. AL1Ac, respectively.

In consideration of these results of the antibiotic sensitivity test and the selection marker genes used for the eukaryotes transformation system, the selection marker genes presented in the following Table 1 were found to be effective in the Labyrinthulomycetes transformation system.

TABLE 1 Tested strain Usable selection marker genes T. aureum Neo^(r), Hyg^(r), Bla^(r) Thraustochytrium sp. Neo^(r), Hyg^(r), Bla^(r), Ble^(r) A. limacinum mh0186 Neo^(r), Hyg^(r), Bla^(r), Ble^(r) Schizochytrium sp. AL1Ac Neo^(r), Hyg^(r) Neo^(r): Neomycin resistant gene, Hyg^(r): Hygromycin resistant gene Bla^(r): Blastcidin resistant gene, Ble^(r): Bleomycin resistant gene

Example 3 Isolation of T. aureum-Derived EF-1α and Ubiquitin Genes, and Isolation of Gene Expression Regulatory Regions

(1) Isolation of T. aureum-Derived EF-1α Gene and Gene Expression Region i. Isolation of T. aureum-Derived EF-1a Gene cDNA Sequence

T. aureum cells cultured in a GY liquid medium were harvested in the logarithmic growth phase to stationary phase by centrifugation at 4° C., 3,500×g for 10 min. The resulting cells were suspended in a sterilized physiological saline, and washed by recentrifugation. The cells were then ground into a powder with a mortar after rapid freezing with liquid nitrogen. Total RNA was extracted from the disrupted cell solution using a Sepasol RNA I Super (nacalai tesque), and mRNA was purified from the total RNA using an Oligotex™-dT30 < Super> mRNA Purification Kit (Takara Bio).

Thereafter, a cDNA library including a synthetic adapter added to the 5′- and 3′-ends was produced using a SMART™ RACE cDNA Amplification Kit (clontech). A single forward degenerate oligonucleotide primer EF-F1 (SEQ ID NO: 1 in the Sequence Listing) was produced based on a known EF-1α conserved sequence using a DNA synthesizer (Applied Biosystems). 3′ RACE performed with these materials confirmed a specific amplification product. The DNA fragments isolated by electrophoresis on a 1% agarose gel were cut out with, for example, a clean cutter, and the DNA was extracted from the agarose gel according to the method described in Non-Patent Document 10. This was followed by the TA cloning of the DNA fragments using a pGEMR-T easy Vector System I (Promega), and the base sequences of these fragments were determined according to the method of Sanger et al. (Non-Patent Document 11). Specifically, the base sequences were determined by the dieterminator technique using a BigDyeR Terminator v3.1 Cyele Sequencing Kit, and a 3130 genetic analyzer (Applied Biosystems). The result that the resulting 980-bp 3′ RACE product (SEQ ID NO: 2 in the Sequence Listing) was highly homologous to the EF-1a genes derived from other organisms strongly suggested that the product was a partial sequence of the T. aureum-derived EF-1a gene.

From this sequence, two reverse oligonucleotide primers EF-1r (SEQ ID NO: 3 in the Sequence Listing) and EF-2r (SEQ ID NO: 4 in the Sequence Listing) were produced, and 5′ RACE was performed using these primers. The result confirmed 5′ RACE products specific to the both. A base sequence analysis found that the former was a 496-bp partial sequence (SEQ ID NO: 5 in the Sequence Listing) of the T. aureum-derived putative EF-1a gene, and the latter a 436-bp (SEQ ID NO: 6 in the Sequence Listing) partial sequence of the T. aureum-derived putative EF-1a gene. There was a complete match with the 3′ RACE product in the overlapping portions.

It was found from these results that the cDNA sequence of the T. aureum-derived putative EF-1a gene was a 1,396-bp sequence (SEQ ID NO: 7 in the Sequence Listing), and that the ORF region was a 1,023-bp region (SEQ ID NO: 9 in the Sequence Listing) encoding 341 amino acid residues (SEQ ID NO: 8 in the Sequence Listing).

ii. Isolation of T. aureum-Derived EF-1a Gene Regulatory Region

T. aureum cells cultured in GY medium were harvested by centrifugation. The resulting cells were suspended in a sterilized physiological saline, and washed by recentrifugation. The cells were then ground into a powder with a mortar after rapid freezing with liquid nitrogen. The genomic DNA was extracted according to the method described in Non-Patent Document 12, and A260/280 was taken to measure the purity and concentration of the extracted genomic DNA.

This was followed by PCR genome walking to isolate the EF-1α gene ORF upstream sequence (promoter) or ORF downstream sequence (terminator), using an LA PCR in vitro Cloning Kit. Note that a reverse oligonucleotide primer r3 (SEQ ID NO: 10 in the Sequence Listing) was used for the amplification of the ORF upstream sequence, and forward oligonucleotide primers EF-t-F1 (SEQ ID NO: 11 in the Sequence Listing) and EF-t-F2 (SEQ ID NO: 12 in the Sequence Listing) were used for the amplification of the ORF downstream sequence. Analysis of the base sequences of the resulting specific amplification products revealed successful isolation of a 615-bp ORF upstream sequence (SEQ ID NO: 13 in the Sequence Listing), and a 1, 414-bp ORF downstream sequence (SEQ ID NO: 14 in the Sequence Listing) of the T. aureum-derived EF-1α gene. In the following, the former is denoted as EF-1α promoter, and the latter EF-1α terminator.

(2) Isolation of T. aureum-Derived Ubiquitin Gene and Gene Expression Region i. Isolation of T. aureum-Derived Ubiquitin Gene cDNA Sequence

3′ RACE was performed with a forward degenerate oligonucleotide primer 2F (SEQ ID NO: 15 in the Sequence Listing) produced from a known ubiquitin gene conserved sequence, using the cDNA library created by using a SMART™ RACE cDNA Amplification Kit (clontech) as a template. Analysis of the base sequence of the resulting amplification product revealed that the product was a 278-bp partial sequence (SEQ ID NO: 16 in the Sequence Listing) of the T. aureum-derived putative ubiquitin gene. Specific amplification products could not be obtained in 5′ RACE, despite use of various oligonucleotide primers under different PCR conditions. This raised the possibility that the high GC-content higher-order structure of the target mRNA might have inhibited the reverse transcription reaction in the cDNA library production.

5′ RACE was thus performed using a 5′ RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Invitrogen), which uses a reverse transcriptase having high heat stability. Note that reverse oligonucleotide primer 1R (SEQ ID NO: 17 in the Sequence Listing) was used for the reverse transcription reaction, and reverse nucleotide primer 2R (SEQ ID NO: 18 in the Sequence Listing) was used for the PCR reaction after the cDNA synthesis. Analysis of the base sequence of the resulting amplification product revealed that the product was a 260-bp partial sequence (SEQ ID NO: 19 in the Sequence Listing) of the T. aureum-derived putative ubiquitin gene, and there was a complete match with the 3′ RACE product in the overlapping portion. The result thus revealed successful isolation of the T. aureum-derived putative ubiquitin gene cDNA sequence.

However, it is known that the ubiquitin gene typically has a repeat structure of the same sequence. It is thus speculated the result did not represent the full-length structure of the gene, but rather revealed the 5′- and 3′-end noncoding regions, and the single sequence forming the repeat structure in the ORF region. Note that the single sequence found in the ORF region of the T. aureum-derived putative ubiquitin gene was found to be a 228-bp sequence (SEQ ID NO: 21 in the Sequence Listing) encoding 76 amino acid residues (SEQ ID NO: 20 in the Sequence Listing).

ii. Isolation of T. aureum-Derived Ubiquitin Gene Regulatory Region

PCR genome walking was performed to isolate a ubiquitin gene ORF upstream sequence (promoter) or an ORF downstream sequence (terminator), using an LA PCR in vitro Cloning Kit. Note that reverse oligonucleotide primers REVERS-U PR-1 (SEQ ID NO: 22 in the Sequence Listing) and REVERS-U PR-2 (SEQ ID NO: 23 in the Sequence Listing) were used for the amplification of the ORF upstream sequence, and forward oligonucleotide primers ubqterminalf1 (SEQ ID NO: 24 in the Sequence Listing) and ter2F (SEQ ID NO: 25 in the Sequence Listing) were used for the amplification of the ORF downstream sequence. Analysis of the base sequences of the specific amplification products revealed successful isolation of a 801-bp ORF upstream sequence (SEQ ID NO: 26 in the Sequence Listing), and a 584-bp ORF downstream sequence (SEQ ID NO: 27 in the Sequence Listing) of the T. aureum-derived ubiquitin gene. In the following, the former will be denoted as ubiquitin promoter, and the latter ubiquitin terminator.

In this manner, the promoters and terminators of the T. aureum-derived house keeping gene EF-1a and the ubiquitin gene were successfully isolated as the gene expression regulatory regions that constantly function in Labyrinthulomycetes.

Example 4 Production of Drug-Resistant Gene Expression Cassette (1) Artificial Synthesis of Neomycin-Resistant Gene (Neo^(r))

Artificial Neo^(r) was synthesized by MediBic according to the codon usage of T. aureum in codon usage database (www.kazusa.or.jp/codon/). The base sequence is represented by SEQ ID NO: 28 in the Sequence Listing, and the encoded amino acid sequence by SEQ ID NO: 29 of the Sequence Listing.

(2) Construction of Neo^(r) Expression Cassette

i. Construction of Neo^(r) Expression Cassette Using EF-1α Promoter and Terminator

A DNA fragment including T. aureum-derived 18S rDNA joined by fusion PCR to the upstream side of a drug-resistant gene (Neo^(r)) expression cassette including EF-1α promoter/artificial Neo^(r)/EF-1α terminator was produced by using the oligonucleotide primers represented by SEQ ID NOS: 30 to 38 of the Sequence Listing, according to the method described in Nippon Nogeikagaku Kaishi, Vol. 77, No. 2 (February, 2003), p. 150-153. PCR reaction was run at a denature temperature of 98° C. for 10 seconds, and the annealing and extension reactions were appropriately adjusted according to the Tm of the primers, and the length of the amplification product.

As a result, T. aureum 18S rDNA, EF-1α promoter, artificial Neo^(r), and EF-1α terminator were successfully joined (4,454 bp; SEQ ID NO: 39 in the Sequence Listing; FIG. 10).

By TA cloning using a pGEM-T easy (Invitrogen), a Labyrinthula-Escherichia coli shuttle vector was constructed that included a Neo^(r) expression cassette with the EF-1α promoter and terminator used as the selection marker for Labyrinthula, and the T. aureum-derived 18S rDNA sequence as a homologous recombination site. In the following, this will be denoted as pEFNeomycin^(r) (FIG. 12).

ii. Construction of Neo^(r) Expression Cassette Using Ubiquitin Promoter and Terminator

The same technique used for the Neo^(r) expression cassette using the EF-1α promoter and terminator was used to join T. aureum 18S rDNA, ubiquitin promoter, artificial Neo^(r), and ubiquitin terminator, using the oligonucleotide primers represented by SEQ ID NOS: 40 to 47 of the Sequence Listing (FIG. 11). The constructed Neo^(r) expression cassette was incorporated by using the NdeI/KpnI site of a pUC18 vector, and a Labyrinthula-Escherichia coli shuttle vector was constructed that included a Neo^(r) expression cassette with the ubiquitin promoter and terminator used as the selection marker for Labyrinthula, and the T. aureum-derived 18S rDNA sequence as a homologous recombination site. In the following, this will be denoted as pUBNeomycin^(r) (FIG. 12).

In this manner, two vectors were constructed: The Labyrinthula-Escherichia coli shuttle vector pEFNeomycin^(r) including the Neo^(r) expression cassette with the EF-1α gene promoter and terminator used as the selection marker for Labyrinthula; and the pUBNeomycin^(r) including the Neo^(r) expression cassette with the ubiquitin gene promoter and terminator. For easy Neo^(r) expression in Labyrinthula, these vectors use the artificially synthesized Neo^(r) whose codons have been optimized by using the T. aureum codon usage as reference. Further, the vectors include the T. aureum-derived 18S rDNA sequence, taking into consideration incorporation into chromosomal DNA by homologous recombination (FIGS. 10 and 11).

Example 5 Gene Introduction Experiment Using Labyrinthula (1) DNAs Used in Gene Introduction Experiment

Gene introduction experiment was conducted using the following four DNAs.

(1) Cyclic vector pUBNeomycin^(r) (2) Cyclic vector pEFNeomycin^(r) (3) Linear Neo^(r) expression cassette adopting ubiquitin promoter and terminator (ub-Neo^(r)) (4) Linear Neo^(r) expression cassette adopting EF-1α promoter and terminator (EF-Neo^(r))

For (3), PCR was performed with an oligonucleotide primer set Ubpro-fug-18s-F (SEQ ID NO: 42 in the Sequence Listing)/KpnterR (SEQ ID NO: 47 in the Sequence Listing), using an LA taq Hot Start Version (Takara Bio), and pUBNeomycin^(r) as a template, and the resulting amplification product was gel purified. For (4), PCR was performed with an oligonucleotide primer set 2F (SEQ ID NO: 32 in the Sequence Listing)/terminator 5R (SEQ ID NO: 33 in the Sequence Listing), using an LA taq Hot Start Version (Takara Bio), and pEFNeomycin^(r) as a template, and the resulting amplification product was gel purified.

(2) Gene Introducing Technique Used for Gene Introduction Experiment

i. Electroporation

Labyrinthulomycetes were cultured in a GY liquid medium to the middle to late stage of the logarithmic growth phase at 25° C., 150 rpm, and the supernatant was removed by centrifugation at 3,500×g, 4° C. for 10 min. The resulting cells were suspended in sterilized 1.75% Sea Life (Marine Tech), and washed by recentrifugation. The cells (5×10⁶) were then suspended in 50 mM sucrose, or in a reagent for gene introduction attached to the equipment used. After applying electrical pulses in different settings, GY liquid medium (1 ml) was immediately added, and the cells were cultured at 25° C. for 12 hours. The culture fluid was then applied to a PDA agar plate medium containing 2 mg/ml G418 (T. aureum, Thraustochytrium sp. ATCC 26185, Schizocytrium sp. AL1Ac) or 0.5 mg/ml G418 (A. limacinum mh0186). After static culturing at 25° C., colony formation of transfectants with the conferred G418 resistance was observed.

ii. Gene Gun Technique

Labyrinthulomycetes were cultured in a GY liquid medium to the middle to late stage of the logarithmic growth phase at 25° C., 150 rpm, and the supernatant was removed by centrifugation at 3,500×g, 4° C. for 10 min. The resulting cells were resuspended in a GY liquid medium in 100 times the concentration of the original culture fluid, and a 20-μl portion of the cell suspension was evenly applied as a thin layer of about a 3-cm diameter on a 5-cm diameter PDA agar plate medium containing or not containing G418. After drying, DNA penetration was performed by using the gene gun technique, using a PDS-1000/He system (Bio-Rad Laboratories). The penetration conditions were investigated by varying the penetration pressure, as follows.

-   -   target distance: 6 cm (fixed)     -   vacuum: 26 inches Hg (fixed)     -   micro carrier size: 0.6 μm (fixed)     -   Rupture disk (penetration pressure): 450, 900, 1100, 1350, and         1,550

In the case of the G418-containing PDA agar plate medium, the cells after the penetration were statically cultured for about 12 hours from the introduction. The static culture was continued after spreading the cells with 100 μl of PDA liquid medium on the PDA agar plate. On the other hand, in the PDA agar plate medium containing no G418, the cells after the penetration were statically cultured for about 12 hours from the introduction, collected, and reapplied to a PDA agar plate medium containing 2 mg/ml or 0.5 mg/ml G418. After static culturing at 25° C., colony formation of transfectants with the conferred G418 resistance was observed.

(3) Acquisition and Evaluation of Transfectant

i. A. limacinum Transfectant

Gene introduction was performed by electroporation under the following conditions

-   -   introduced DNA: pUBNeomycin^(r) and ub-Neo^(r)     -   gene introducing technique: electroporation     -   cell suspension buffer: 50 mM sucrose     -   gene introducing apparatus: Gene Pulser (Bio-Rad Laboratories)         with a 1-mm gap cuvette     -   pulse settings: 50 μF/50 Ω/0.75 kV, single application

In samples using the linear DNA ub-Neo^(r), G418-resistant colonies were observed at the efficiency as high as 2.4×10⁰ cfu/μq DNA. On the other hand, in samples using the cyclic DNA pUBNeomycin^(r), no colony formation was observed, regardless of multiple introductions.

A comparative examination of introduction efficiency was made using a gene introducing apparatus. Introduction tests were conducted using a Microporator MP-100 (AR Brown) or a Nucleofector™ (amaxa) with the attached condition search kit. While no single colony was formed with the Microporator MP-100, the Nucleofector used with the attached cell suspension buffer Nucleofector R solution L produced transfectants with good reproducibility at the efficiency as high as 9.5×10⁰ cfu/μg DNA.

Then, pulse settings were examined with the Nucleofector R solution L, using a Gene Pulser (Bio-Rad Laboratories). It was found as a result that transfectants could be obtained with good reproducibility at the efficiency as high as 1.2×10¹ cfu/μg DNA by double application under the following conditions: capacitance 50 μF, electrical resistance 50Ω, and electric field intensity 0.75 kV. The results are summarized in Table 2 below.

TABLE 2 Gene Introduction Gene introducing reagent (cell Pulse introduction apparatus suspension buffer) settings efficiency Gene Pulser 50 mM sucrose 50 μF/50 up to 2.4 × 10⁰ Ω/0.75 kV, cfu/μg DNA single application Microporater Attached buffer Conditions — MP-100 specified in the manual Nucleofector ™ Attached buffer Conditions up to 9.5 × 10⁰ (Necleofector ® specified in cfu/μg DNA solution L) the manual Gene Pulser Necleofector ® 50 μF/50 up to 1.2 × 10¹ solution L Ω/0.75 kV, cfu/μg DNA double application ii. Evaluation of A. limacinum Transfectant Using G418-Resistance as Index

The transfectants were cultured in 0.5 mg/ml G418-containing GY liquid medium. The wild-type strain was cultured in GY liquid medium containing no G418. The culture fluids of these strains were spotted in 10-μl portions on PDA agar plate media containing G418 (0, 0.2, 0.5, 1, 2, 4 mg/ml), and growth on the agar plate media was observed after culturing the cells at 25° C. for 2 days. It was found as a result that the proliferation was inhibited at 0.2 mg/ml G418 in the wild-type strain, whereas the transfectants proliferated even in the presence of 4 mg/ml G418 (FIG. 13A). Further, there was no change in G418 resistance, and proliferation was observed even at a G418 concentration of 32 mg/ml in a similar experiment conducted with PDA agar plate media containing G418 (0, 2, 4, 8, 16, 32 mg/ml) after subculturing the transfectants five times in a GY liquid medium containing no G418 (FIG. 13B). These results using the G418 resistance as an index confirmed that the conferred character was stable.

iii. Morphology Comparison of A. limacinum Transfectant and Wild-Type Strain

It was confirmed by microscopy (FIG. 14A) and by confocal laser microscope observation after staining the oil globules in the cells with nile red (FIG. 14B) that there was no morphological change between the wild-type strain and the transfectants. Further, 18S rDNA analysis confirmed that the transfectants were A. limacinum.

iv. Evaluation of A. limacinum Transfectant by PCR Using Genomic DNA as Template

The transfectants were cultured in 0.5 mg/ml G418-containing GY liquid medium. The wild-type strain was cultured in GY liquid medium containing no G418. Genomic DNA was extracted from the cells of each strain by using an ISOPLANT (nacalai tesque). Using the genomic DNA as a template, Neo^(r) was amplified by PCR using an LA taq Hot Start Version (Takara Bio). The oligonucleotide primers ubproG418fus2F (SEQ ID NO: 45 in the Sequence Listing)/G418ubtersus3R (SEQ ID NO: 46 in the Sequence Listing) were used (PCR cycles: 94° C. 2 min/98° C. 10 sec, 68° C. 1 min, 72° C., 30 cycles/4° C.).

As a result, specific Neo^(r) amplification, not found in the wild-type strain, was observed in the transfectants (FIG. 15). The result thus suggested that the introduced ub-Neo^(r) was incorporated in the chromosomal DNA.

v. Evaluation of A. limacinum Transfectant by Southern Blotting

Genomic DNAs (2 μg) of the A. limacinum transfectants and the wild-type strain extracted according to an ordinary method were digested with various restriction enzymes at 37° C. for 16 hours, and Southern blotting was performed according to the DIG Manual, 8th, Roche, using a DIG-labeled Neo^(r) as a probe.

As a result, a Neo^(r) band was detected, as shown in FIG. 16A. This suggested that the ub-Neo^(r) by the introduced ubiquitin promoter and terminator had been incorporated in the chromosomal DNA. Further, the result that the five transfectant bands digested with the same enzyme (PstI) had different molecular weights suggested that the introduced DNA fragment was randomly incorporated in the chromosomal DNA (FIG. 16B).

vi. Evaluation of A. limacinum Transfectant by RT-PCR

Total RNA was extracted from the cells of the A. limacinum transfectants and the wild-type strain using a Sepasol RNA I super (nacalai tesque). After cleaning the total RNA using an RNeasy plus mini kit (QIAGEN), a reaction was run at 37° C. for 1 hour by using a Cloned DNase I (Takara Bio) according to the attached manual to degrade the contaminated genomic DNA. This was followed by a reverse transcription reaction using a PrimeScript Reverse Transcriptase (Takara Bio) to synthesize cDNA by reverse transcription reaction. The cDNA was used as a template to amplify Neo^(r) by PCR using an LA taq Hot Start Version (Takara Bio). The oligonucleotide primers ubproG418fus2F (SEQ ID NO: 45 in the Sequence Listing)/G418ubtersus3R (SEQ ID NO: 46 in the Sequence Listing) were used (PCR cycles: 94° C. 2 min/98° C. 10 sec, 68° C. 1 min, 72° C., 30 cycles/4° C.).

As a result, Neo^(r) amplification products were confirmed in the transfectants (FIG. 17, lanes 1 to 5). The result that amplification products were not observed in a PCR using the total RNA as a template (FIG. 17, lanes 8 to 13) suggested that the products observed in lanes 1 to 5 were not genomic DNA contamination, but originated in the Neo^(r) mRNA reverse transcripts (Neo^(r) cDNA). It was therefore found that the ub-Neo^(r) incorporated in the chromosomal DNA was subject to transcription into mRNA.

vii. Acquisition of T. aureum Transfectant

Two types of DNAs, pUBNeomycin^(r) and ub-Neo^(r), were used as the introduced DNAs. After investigating various conditions, it was found that no transfectants could be obtained by electroporation. With the gene gun technique, however, it was possible to acquire transfectants with the conferred G418 resistance. The gene introduction efficiency was the highest at a penetration pressure of 1,100 psi, specifically as high as 1.9×10² cfu/μg DNA in the case of ub-Neo^(r). The gene introduction efficiency was as high as 1.4×10¹ cfu/μg DNA for the pUBNeomycin^(r), showing that the introduction efficiency was about 14 times higher in the random integration introducing the liner DNA than in the introduction of the cyclic DNA using the 18S rDNA sequence as a homologous recombination site. It was also found that the transfectants maintained the G418 resistance even after being subcultured five times in a GY liquid medium containing no G418.

viii. Morphology Comparison of T. aureum Transfectant and Wild-Type Strain

Confocal laser microscope observation after staining the oil globules of the cells with nile red (FIG. 18) confirmed no morphological change between the wild-type strain and the transfectants. Further, 18S rDNA analysis confirmed that the transfectants were T. aureum.

ix. Evaluation of T. aureum Transfectant by PCR Using Genomic DNA as Template and by Southern Blotting

As with the case of the A. limacinum transfectants, random incorporation of ub-Neo^(r) in the chromosomal DNA was confirmed by PCR using the genomic DNA as a template (FIG. 19A), and by Southern blotting detecting Neo^(r) (FIG. 19B).

x. Evaluation of T. aureum Transfectant by RT-PCR

As with the case of the A. limacinum transfectants, it was found that the ub-Neo^(r) incorporated in the chromosomal DNA was subject to transcription into mRNA (FIG. 20).

xi. Acquisition of Thraustochytrium sp. ATCC 26185 Transfectant

A linear Neo^(r) expression cassette adopting EF-1α promoter and terminator (EF-Neo^(r)) was used as the introduced DNA. After investigating various conditions, transfectants were obtained by electroporation at a very low gene introduction efficiency (10¹ cfu/μg DNA or less). It was also found that the transfectants maintained the G418 resistance even after being subcultured five times in a GY liquid medium containing no G418.

xii. Evaluation of Thraustochytrium sp. ATCC 26185 Transfectant by PCR Using Genomic DNA as Template and by Southern Blotting

As with the case of the A. limacinum transfectants, random incorporation of EF-Neo^(r) into the chromosomal DNA was confirmed by PCR using the genomic DNA as a template (FIG. 21A, B), and by Southern blotting detecting Neo^(r) (FIG. 21C). However, a presence of partial defects in the terminator region was suggested in one of the three transfectants analyzed (Transfectant 2; FIG. 21B, lane7).

xiii. Evaluation of Thraustochytrium sp. ATCC 26185 Transfectant by RT-PCR

It was found that the EF-Neo^(r) incorporated in the chromosomal DNA was subject to transcription into mRNA (FIG. 22A, B), including the Transfectant 2 in which partial defects in the terminator region were suggested (FIG. 22, lane 14).

xiv. Acquisition of Schizochytrium sp. AL1Ac Transfectant

ub-Neo^(r) was used as the introduced DNA. Despite investigation of various conditions, no transfectants could be obtained by electroporation. However, with the gene gun technique examined under different conditions, it was possible to obtain transfectants at a penetration pressure of 1,100 psi, even though the gene introduction efficiency was very low (10⁻¹ cfu/μg DNA or less). It was also found that the transfectants maintained the G418 resistance even after being subcultured five times in a GY liquid medium containing no G418.

xv. Evaluation of Schizochytrium sp. AL1Ac Transfectant by PCR Using Genomic DNA as Template

As with the case of the A. limacinum transfectants, incorporation of the introduced DNA fragments in the chromosomal DNA was strongly suggested by PCR using the genomic DNA as a template (FIG. 23).

As the results of these gene introduction experiments demonstrate, it became possible to obtain transfectants of all four strains of Labyrinthula by random integration using the linear DNA, and electroporation or gene gun technique (Table 3).

TABLE 3 Gene introduction Gene introduction Tested strain method efficiency A. limacinum mh0186 Electroporation up to 1.2 × 10¹ cfu/μg DNA T. aureum Gene gun up to 1.9 × 10² cfu/μg DNA Thraustochytrium sp. Electroperation up to 10⁰ cfu/μg DNA Schizochytrium sp. AL1Ac Gene gun up to 10⁰ cfu/μg DNA

The G418 resistance of the transfectants was stable, suggesting that the introduced DNA was randomly incorporated in the chromosomal DNA, as evaluated by PCR using the genomic DNA as a template, or by Southern blotting analysis.

Example 6

Expression of Foreign Protein in Aurantiochytrium limacinum mh0186 and Thraustochytrium aureum ATCC 34304 by Transformation

(1) Expression of Aequorea green fluorescent protein (GFP) i. Incorporation of GFP Gene into mh0186 Genomic DNA

The ubiquitin gene-derived promoter and terminator regions derived from Thraustochytrium aureum ATCC 34304 (obtained from American type culture collection), and Enhanced GFP gene (Clontech) were amplified by PCR using a PrimeSTAR GC polymerase kit (Takara Bio) (PCR cycles: 94° C. 2 min/94° C. 1 min, 62° C. 30 sec, 72° C. 1 min, 30 cycles/4° C.). The promoter region and the GFP gene were joined by fusion PCR using a PrimeSTAR GC polymerase kit (Takara Bio) (PCR cycles: 94° C. 2 min/94° C. 1 min, 62° C. 30 sec, 72° C. 2 min, 30 cycles/4° C.). By using this as a template, the promoter region, the GFP gene, and the terminator region were joined by fusion PCR with a PrimeSTAR GC polymerase kit (Takara Bio) (PCR cycles: 94° C. 2 min/94° C. 1 min, 62° C. 30 sec, 72° C. 3 min, 30 cycles/4° C.). The joined DNA fragment was then incorporated in a pGEM-T Easy vector (Promega). By using this plasmid as a template, a KpnI site was added to the both ends of the GFP gene cassette by PCR performed with primers Ub-pro-F1 (SEQ ID NO: 48 in the Sequence Listing) and Ub-term-R2 (SEQ ID NO: 49 in the Sequence Listing), using a PrimeSTAR GC polymerase kit (Takara Bio). The resulting cassette was then incorporated at the KpnI site (immediately following the terminator region) of a pUC18 vector including an artificially synthesized neomycin-resistant gene cassette (ubiquitin gene-derived promoter and terminator regions) to produce a GFP gene/neomycin-resistant gene expression cassette. The vector including the GFP gene/neomycin-resistant gene expression cassette was named pNeoGFP.

The GFP gene/neomycin-resistant gene expression cassette was amplified with primers Ub18Spro-F2 (SEQ ID NO: 50 in the Sequence Listing) and pUC18-R (SEQ ID NO: 51 in the Sequence Listing), using a PrimeSTAR GC polymerase kit (Takara Bio). After purification, the purified DNA fragment (5 μg) was introduced into the mh0186 strain. This was performed by following the gene introduction procedure in which cells cultured in a 200-ml GY liquid medium for 3 days were suspended in 0.3 M sorbitol (Wako Pure Chemical Industries, Ltd.) or in Nucleofector Solution L (lonza) used as a final cell suspension, and then subjected to electroporation under 0.75 kV, 50Ω, 50 μF conditions using a GENE PULSER® II (Bio-Rad Laboratories). The DNA fragments (0.625 μg) purified in a similar fashion were also introduced into T. aureum cultured in a 200-ml GY liquid medium for 5 days, by using the gene gun technique with a Standard Pressure Kit (Bio-Rad Laboratories) and a PDS-1000/He system (Bio-Rad Laboratories). The DNA was introduced by penetrating the cells applied onto a PDA agar plate medium (containing 2 mg/ml G418), under the following conditions: 0.6-micron gold particles, target distance 6 cm, vacuum 26 mmHg, Rupture disk 1,100 PSI.

For the mh0186 strain, genomic DNA was extracted from cells cultured in a 100-ml GY liquid medium (containing 0.5 mg/ml G418) for 3 days. In the case of T. aureum, genomic DNA was extracted from cells cultured in a 100-ml GY liquid medium (containing 2 mg/ml G418) for 7 days. The purity and the concentration of the extracted genomic DNA were measured by measuring A260/280 using an Ultrospec 3000 (Amersham Pharmacia Biotech). By using the extracted genomic DNA as a template, PCR was performed with primers 3F (SEQ ID NO: 52 in the Sequence Listing), 4R (SEQ ID NO: 53 in the Sequence Listing), Ub-GFP-F (SEQ ID NO: 54 in the Sequence Listing), and UB-GFP-R (SEQ ID NO: 55 in the Sequence Listing), using an LA Taq HS polymerase Kit (Takara Bio) (PCR cycles: 98° C. 2 min/98° C. 20 sec, 60° C. 30 sec, 72° C. 1 min, 30 cycles/4° C.).

Fusion PCR performed with the chimeric primers presented in Table 4 joined all the GFP gene, ubiquitin gene promoter region, and ubiquitin gene terminator region. The resulting fragment was incorporated at the KpnI site (immediately following the terminator region) of a pUC18 vector (Takara Bio) including an artificially synthesized neomycin-resistant gene cassette (ubiquitin gene-derived promoter and terminator regions) to produce a GFP gene/neomycin-resistant gene expression cassette (FIG. 24). Introducing the GFP gene/neomycin-resistant gene expression cassette into the A. limacinum mh0186 strain and T. aureum produced transfectants. These transfectants were subjected to a PCR using the genomic DNA as a template, and the result confirmed that the GFP gene and the neomycin-resistant gene were successfully incorporated into the genomic DNA of the GFP gene/neomycin-resistant gene expression cassette transfectants (FIG. 25).

TABLE 4 Name Sequence Direction Ub18Spro-F2 5′-AGAGGAAGGTGAAGTCGTAACAAGGCGTTAGA-3′ Forward Ub-pro-F1 5′-TCGGTACCCGTTAGAACGCGTAATACGAC-3′ Forward Ub-pro-R1 5′-TCCTCGCCCTTGCTCACCATGTTGGCTAGTGTTGCTTAGGT-3′ Reverse Ub-GFP-F 5′-ACCTAAGCAACACTAGCCAACATGGTGAGCAAGGGCGAGGA-3′ Forward Ub-GFP-R 5′-AGCACATACTACAGATAGCTTAGTTTTACTTGTACAGCTCGTCCA-3′ Reverse Ub-term-F1 5′-TGGACGAGCTGTACAAGTAAAACTAAGCTATCTGTAGTATGTGCT-3′ Forward Ub-term-R1 5′-ATCTAGAACCGCGTAATACGACTCACTATAGGGAGAC-3′ Reverse Ub-term-R2 5′-TCGGTACCACCGCGTAATACGACTCACTATAGGGAGACTGCAGTT-3′ Reverse pUC18-R 5′-AACAGCTATGACCATGATTACGAATTCGAGCTCGG-3′ Reverse Ub-pro-F1 and Ub-term-R2 have KpnI site in the sequence (underlined). ii. GFP mRNA Expression

For the mh0186 strain, total RNA was extracted from a main cell culture incubated in a 100-ml GY liquid medium (containing 0.5 mg/ml G418) for 3 days. In the case of T. aureum, total RNA was extracted from cells cultured in a 100-ml GY liquid medium (containing 2 mg/ml G418) for 7 days. Sepasol RNAI Super (nacalai tesque) was used for the extraction. The total RNA was cleaned by using an RNeasy Mini Kit (QIAGEN). The purity of the total RNA was increased by a DNase treatment using a Cloned DNaseI (Takara Bio), and the purity and the concentration of the purified total RNA were measured by measuring A260/280 using an Ultrospec 3000 (Amersham Pharmacia Biotech). cDNA was produced from the purified total RNA using a PrimeScript™ Reverse Transcriptase (Takara Bio). By using the cDNA as a template, a PCR was performed with primers 3F (SEQ ID NO: 52 in the Sequence Listing), 4R (SEQ ID NO: 53 in the Sequence Listing), Ub-GFP-F (SEQ ID NO: 54 in the Sequence Listing), and UB-GFP-R (SEQ ID NO: 55 in the Sequence Listing), using an LA Taq HS polymerase kit (Takara Bio) (PCR cycles: 98° C. 2 min/98° C. 20 sec, 60° C. 30 sec, 72° C. 1 min, 30 cycles/4° C.).

The result indicated that the incorporated GFP gene and neomycin-resistant gene were subject to transcription into mRNA (FIG. 26).

iii. GFP Expression

For the mh0186 strain, cells cultured in a 3-ml GY liquid medium (containing 0.5 mg/ml G418) for 3 days were harvested by centrifugation at room temperature, 3,500×g for 10 min. In the case of T. aureum, cells (1 ml) cultured in a 100-ml GY liquid medium (containing 2 mg/ml G418) for 7 days were harvested by centrifugation performed under the same conditions. The harvested cells were washed twice with a 500-μl sterilized SEA LIFE, observed under a confocal laser microscope (ECLIPSE TE2000-U; Nikon; 40×60 magnification, oil-immersion lens, excitation light Ar laser 488 nm), and imaged by using EZ-C1 software (Nikon).

Confocal laser microscopy showed GFP fluorescence in the GFP gene/neomycin-resistant gene expression cassette transfectants, but not in the wild type (FIG. 27).

(2) Pinguiochrysis Δ12 Desaturase Expression

i. Cloning of Δ12 Desaturase

Pinguiochrysis pyriformis MBIC 10872 (obtained from Marine Biotechnology Institute Culture collection) was cultured in ESM medium (produced according to the method described in the medium list of the NIES collection), and cells at the late stage of the logarithmic growth phase were harvested by centrifugation at 4° C., 6,000×g for 15 min. The harvested cells were frozen by liquid nitrogen, and total RNA was extracted by using the phenol/SDS/LiCl technique (1). Then, poly (A) +RNA was purified from the total RNA, using a mRNA Purification Kit (GE healthcare Bio-sciences). Single-stranded cDNA was then produced from the purified poly (A) +RNA, using a Ready-To-Go T-Primed First-Strand Kit (GE healthcare Bio-sciences). By using the cDNA as a template, a PCR was performed with primers F1 (SEQ ID NO: 56 in the Sequence Listing) and R1 (SEQ ID NO: 57 in the Sequence Listing) produced based on a known conserved sequence of Δ12 desaturase, using an Advantage™ 2 PCR Kit (Clontech) (PCR cycles: 95° C. 30 sec, 50° C. 30 sec, 68° C. 2 min, 40 cycles/4° C.). The amplified PCR product was incorporated in a pGEM-T easy vector (Promega), and introduced into competent cells DH5a (Toyobo) by electroporation. By using the extracted transfectant plasmid as a template, the base sequence was analyzed by sequencing using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). A P. pyriformis cDNA library was constructed using a Lambda cDNA Library Construction Kits (Stratagene). Screening of positive clones was performed by plaque hybridization using an ECL Direct Nucleic Acid Labeling and Detection System (GE healthcare Bio-sciences). As to the incubation conditions with the probe, the clones were incubated at 42° C. for 3 hours with a labeled probe added in an 8 ng/ml concentration, and washed twice at 55° C. for 10 min (primary washing with no urea), and twice at room temperature for 5 min (secondary washing with no urea). As the probe, a 314-bp cDNA fragment amplified by a PCR with primers SP1/F (SEQ ID NO: 58 in the Sequence Listing) and SP1/R (SEQ ID NO: 59 in the Sequence Listing) using an Advantage™ 2 PCR Kit (Clontech) was used (PCR cycles: 94° C. 3 min/94° C. 30 sec, 56° C. 30 sec, 68° C. 1 min, 35 cycles/4° C.). A plasmid containing the acquired partial sequence was used as a template in the PCR. After several screenings, the positive clones were transferred from the λ phage to a pBluescript (Stratagene) using an ExAssist helper phage (Stratagene).

As a result, a 515-bp putative Δ12 desaturase gene partial sequence was successfully amplified. Screening of positive clones including the full length of the target gene by plaque hybridization using the acquired DNA fragment as a probe successfully isolated seven positive clones from 5.5×10⁶ clones. Analyses of these sequences suggested that the acquired gene was a gene containing a 1,314-bp ORF encoding 437 amino acids.

ii. Alignment with Δ12 Desaturases Derived from Other Organisms

Multiple alignment analysis was performed for the amino acid sequences of P. pyriformis-, fungus-, and protozoa-derived Δ12 desaturases, using ClustalW 1.81 and ESPript 2.2 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi).

It was found as a result that the amino acid sequence of the acquired gene had high homology with the amino acid sequences of the Δ12 desaturase genes derived from other organisms (FIG. 28). Further, the putative amino acid sequence of the acquired gene conserved three histidine boxes commonly conserved in desaturase (FIG. 28).

iii. Phylogenetic Analysis

A molecular phylogenetic tree of Δ12 desaturases and Δ12/Δ15 desaturases, including the P. pyriformis-derived Δ12 desaturase, was created by using the maximum-likelihood method with a MOLPHY version 2.3 computer program package (Non-Patent Document 13). First, multiple alignment was performed with ClustalW 1.81 for all amino acid sequences. After removing the uncertain portions, a search for a maximum-likelihood phylogenetic tree was made, using the phylogenetic tree by the neighbor-joining method (2) as the initial phylogenetic tree.

As a result, the acquired putative Δ12 desaturase, and the Δ12 desaturases and Δ12/Δ15 desaturases derived from other organisms were classified into three lineage groups: a fungal & nematode Δ12 desaturase group, a plant Δ12 desaturase group, and a cyanobacterial and chloroplast-localized plant Δ12 desaturase group. The acquired putative Δ12 desaturase was classified into the fungal & nematode Δ12 desaturase group, showing that the Saprolegnia diclina-derived Δ12 desaturase was the closest relative (FIG. 29).

iv. Expression of Δ12 Desaturase in Yeast

By using a plasmid containing the full length of the P. pyriformis-derived Δ12 desaturase gene as a template, a PCR was performed with primers Pry-F (SEQ ID NO: 60 in the Sequence Listing) and Pyr-R (SEQ ID NO: 61 in the Sequence Listing), using a PrimeSTAR GC polymerase kit (TakaraBio). The PCR added a HindIII restriction enzyme site and an XbaI restriction enzyme site at the both ends. The amplified fragments were incorporated in a pGEM-T-Easy vector (Promega), and sequence analysis was performed. The Δ12 desaturase gene was cut out by HindIII/XbaI treatment from a plasmid that did not have amplification error, and incorporated into a yeast vector pYES2/CT (Invitrogen) subjected to the same restriction enzyme treatment. As a result, a Δ12 desaturase gene expression vector pYpΔ12Des was constructed. The Δ12 desaturase gene expression vector pYpΔ12Des and the pYES2/CT were introduced into a budding yeast Saccharomyces cerevisiae by using the lithium acetate method, according to the methods described in Current Protocols in Molecular Biology, Unit 13 (Ausubel et al., 1994) and in Guide to Yeast Genetics and Molecular Biology (Gutherie and Fink, 1991), and the yeasts were screened for transfectants. The transfectants (pYpΔ12Des introduced strain and mock introduced strain) were cultured according to the method of Qiu et al. (Qiu, X., et al. J. Biol. Chem. (2001) 276, 31561-6), and the extraction and methylesterification of the yeast-derived fatty acids were performed. A gas chromatograph GC-2014 (Shimadzu Corporation) was used for GC analysis, which was performed under the following conditions. Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.), column temperature: 150° C.-4 (5° C./min)-4 220° C. (10 min), carrier gas: He (1.3 mL/min). GC-17A and GCMS-QP-5000 (Shimadzu Corporation) were used for GC-MS analysis, which was performed under the following conditions. DB-1 capillary column (0.25 mmi.d.×30 m, film thickness 0.25 μm; Agilent), column temperature 160° C.→(4° C./min)→260° C., injector port temperature 250° C. For peaks that caused troubles in the analyses, the fatty acids were analyzed after picolinyl esterification, using the same apparatuses and columns under the temperature condition 240° C.→(2.5° C./min)-260° C. (15 min)→(2.5° C./min)→280° C.

In order to verify that the Δ12 desaturase was encoded by the acquired gene, an expression vector was constructed, and an expression experiment was conducted using a budding yeast S. cerevisiae (Invitrogen) as a host. A GC analysis of the fatty acid compositions of the pYpΔ12Des introduced strain and the pYES2/CT introduced strain confirmed a new peak in the pYpΔ12Des introduced strain at a position corresponding to the retention time of linoleic acid, but not in the mock control (FIG. 30). A GC-MS analysis of this new peak revealed that the molecular weight and the fragment pattern coincide with those of the sample linoleic acid methyl ester (FIG. 31). The conversion efficiency from oleic acid to linoleic acid was 23.5±1.23%, as calculated according to the following equation.

Conversion efficiency (%)=product (%)/(product (%)+substrate (%))×100

No activity for other fatty acids was observed (Table 5).

TABLE 5 All foreign substrates were added to make the final concentration 40 μM. Substrate (%) and product (%) are the percentage with respect to the total fatty acid (GC peak area). Conversion efficiency (%) = 100 × ([product]/[product + substrate]). All values are mean values ± standard deviation. n = 3 Conversion Substrate Substrate (%) Product Product (%) efficiency (%) Mock 18:1^(Δ9a) 29.6 ± 1.15 18:2^(Δ9,) ¹² ND^(c) 0 16:1^(Δ9a) 47.04 ± 0.62  16:2^(Δ9,) ¹² ND^(c) 0 pYpΔ12des 14:1^(Δ9b) 3.99 ± 0.38 14:2^(Δ9,) ¹² ND^(c) 16:1^(Δ9a) 45.8 ± 0.80 16:2^(Δ9,) ¹² ND^(c) 18:1^(Δ9a) 21.3 ± 0.27 18:2^(Δ9,) ¹² 6.56 ± 0.49 23.5 ± 1.23 18:1^(transΔ9b) 7.60 ± 2.23 18:2^(transΔ9, 12) ND^(c) 18:2^(Δ9, 12b) 18.5 ± 0.30 18:3^(Δ9, 12, 15) ND^(c) 18:3^(Δ6, 9, 12b) 16.3 ± 1.32 18:4^(Δ6, 9, 12, 15) ND^(c) 20:3^(Δ8, 11, 14b) 18.8 ± 0.31 20:4^(Δ8, 11, 14, 17) ND^(c) 20:4^(Δ5, 8, 11, 14b) 26.8 ± 0.75 20:5^(Δ5, 8, 11, 14, 17) ND^(c) 22:4^(Δ7,) ^(10, 13, 16b) 4.21 ± 0.16 22:5^(Δ7, 10, 13, 16, 19) ND^(c) ^(a)Endogenous fatty acid ^(b)Exogenous fatty acid ^(c)ND, below detection limit v. Incorporation of Δ12 Desaturase Gene into mh0186 Genomic DNA

First, ubiquitin gene-derived promoter and terminator regions, and the Δ12 desaturase gene were amplified by PCR using a PrimeSTAR GC polymerase kit (Takara Bio) (PCR cycles: 94° C. 2 min/94° C. 1 min, 62° C. 30 sec, 72° C. 1.5 min, 30 cycles/4° C.) The promoter region and the Δ12 desaturase gene were then joined by fusion PCR using a PrimeSTAR GC polymerase kit (Takara Bio) (PCR cycles: 94° C. 2 min/94° C. 1 min, 62° C. 30 sec, 72° C. 2.5 min, 30 cycles/4° C.). By using this as a template, the promoter region, the GFP gene, and the terminator region were joined by fusion PCR using a PrimeSTAR GC polymerase kit (Takara Bio) (PCR cycles: 94° C. 2 min/94° C. 1 min, 62° C. 30 sec, 72° C. 3 min, 30 cycles/4° C.). The joined DNA fragment was then incorporated into a pGEM-T Easy vector (Promega). By using this plasmid as a template, a single-base mutation was introduced at the KpnI site in the Δ12 desaturase gene sequence using a PrimeSTAR MAX DNA polymerase (Takara Bio). The joined fragment was cut out by KpnI treatment, and incorporated at the KpnI site of a pUC18 vector (Takara Bio) including an artificially synthesized neomycin-resistant gene cassette (EF1-α gene-derived promoter region and terminator region are joined at the both ends). The vector including the Δ12 desaturase gene/neomycin-resistant gene expression cassette was named pNeoDes12. The sequences of the PCR primers used are presented in Table 6. The Δ12 desaturase gene/neomycin-resistant gene expression cassette was amplified with primers 2F (SEQ ID NO: 62 in the Sequence Listing) and pUC18-R (SEQ ID NO: 51 in the Sequence Listing), using a PrimeSTAR GC polymerase kit, and purified. After purification, the purified DNA fragment (5 μg) was introduced into the mh0186 strain as in (1)-1. Nucleofector Solution L (lonza) was used as a final cell suspension. Genomic DNA was extracted from a main cell culture incubated in a 100-ml GY liquid medium (containing 0.5 or 2 mg/ml G418) for 3 days, and the purity and the concentration of the extracted genomic DNA were measured by measuring A260/280 using an Ultrospec 3000 (Amersham Pharmacia Biotech). By using the extracted genomic DNA as a template, a PCR was performed with primers 3F (SEQ ID NO: 52 in the Sequence Listing), 4R (SEQ ID NO: 53 in the Sequence Listing), ub pro-Δ12d-F (SEQ ID NO: 63 in the Sequence Listing), and ub term-Δ12d-R (SEQ ID NO: 64 in the Sequence Listing), using an LA Taq HS polymerase Kit (Takara Bio) (PCR cycles: 98° C. 2 min/98° C. 20 sec, 60° C. 30 sec, 72° C. 1.5 min, 30 cycles/4° C.).

Fusion PCR using the chimeric primers presented in Table 6 successfully joined all the Δ12 desaturase gene, ubiquitin gene promoter region, and ubiquitin gene terminator region. The joined fragment was incorporated at the KpnI site of a pUC18 vector including an artificially synthesized neomycin-resistant gene cassette (EF1-α gene-derived promoter and terminator regions) to produce a Δ12 desaturase gene/neomycin-resistant gene expression cassette (FIG. 32). Introducing the Δ12 desaturase gene/neomycin-resistant gene expression cassette into the mh0186 strain successfully produced transfectants. These transfectants were subjected to a PCR using the genomic DNA as a template, and the result confirmed that the Δ12 desaturase gene and the neomycin-resistant gene were successfully incorporated in the genomic DNA of the Δ12 desaturase gene/neomycin-resistant gene expression cassette transfectants (FIG. 33).

TABLE 6 Name Sequence Direction 18S 5′-CGAATATTCCTGGTTGATCCTGCCAGTAGT-3′ Forward 1R 5′-GTAACGGCTTTTTTTGAATTGCAGGTTCACTACGGAAACCTTGTTA-3′ Reverse 2F 5′-GGTTTCCGTAGTGAACCTGCAATTCAAAAAAAGCCGTTACTCACAT-3′ Forward 3R 5′-AAGGCCGTCCTGTTCAATCATCTAGCCTTCCTTTGCCGCTGCTTGC Reverse T-3′ 3F 5′-CAGCGGCAAAGGAAGGCTAGATGATTGAACAGGACGGCCTTCACGC-3′ Forward 4R 5′-GCGCATAGCCGGCGCGGATCTCAAAAGAACTCGTCCAGGAGGCGGT-3′ Reverse 4F 5′-TCCTGGACGAGTTCTTTTGAGATCCGCGCCGGCTATGCGCCCGTGC-3′ Forward 5R 5′-CACTGCAGCGAAAGACGGGCCGTAAGGACG-3′ Reverse Ub-pro-F1 5′-TCGGTACCCGTTAGAACGCGTAATACGAC-3′ Forward ub-pro-D12d- 5′-AGGTTTCCTCCACGACCCATGTTGGCTAGTGTTGCTTAGGTCGCT-3′ Reverse R ub-pro-D12d- 5′-CCTAAGCAACACTAGCCAACATGGGTCGTGGAGGAAACCTCTCCA-3′ Forward F ub 5′-ATACTACAGATAGCTTAGTTTTAGTCGTGCGCCTTGTAGAACACA-3′ Reverse term-D12d-R ub 5′-TCTACAAGGCGCACGACTAAAACTAAGCTATCTGTAGTATGTGCT-3′ Forward D12d-term-F Ub-term-R2 5′-TCGGTACCACCGCGTAATACGACTCACTATAGGGAGACTGCAGTT-3′ Reverse pUC18-R 5′-AACAGCTATGACCATGATTACGAATTCGAGCTCGG-3′ Reverse D12d-F2 5′-CGCGGTGGG

ACCGGTGTCTGGGTCATCGC-3′ Forward D12d-R2 5′-ACACCGGT

CCCACCGCGCCCTGCCAGAA-3′ Reverse 18S and 5R has SspI site or PstI site in the sequence (underlined). Ub-pro-F1 and Ub-term-R2 has KpnI site in the sequence (underlined). Bold italicized letters in the D12d-F2 and D12d-R2 sequences indicate mutated bases. vi. Incorporation of Δ12 Desaturase Gene in T. aureum. Genomic DNA

The Δ12 desaturase gene/neomycin-resistant gene expression cassette was amplified with primers 2F (SEQ ID NO: 62 in the Sequence Listing) and pUC18-R (SEQ ID NO: 51 in the Sequence Listing), using a PrimeSTAR GC polymerase kit, and purified. After purification, the purified DNA fragment (0.625 μg) was introduced into cells cultured in a 200-ml GY liquid medium for 5 days, using the gene gun technique with a Standard Pressure Kit (Bio-Rad Laboratories) and a PDS-1000/He system (Bio-Rad Laboratories). The DNA was introduced by penetrating the cells applied onto a PDA agar plate medium (containing 2 mg/ml G418), under the following conditions: 0.6-micron gold particles, target distance 6 cm, vacuum 26 mmHg, rupture disk 1,100 PSI. Genomic DNA was extracted from cells cultured in a 100-ml GY liquid medium (containing 2 mg/ml G418) for 7 days, and the purity and the concentration of the extracted genomic DNA were measured by measuring A260/280 using an Ultrospec 3000 (Amersham Pharmacia Biotech). By using the extracted genomic DNA as a template, a PCR was performed with primers 3F (SEQ ID NO: 52 in the Sequence Listing), 4R (SEQ ID NO: 53 in the Sequence Listing), ub pro-D12d-F (SEQ ID NO: 63 in the Sequence Listing), and ub term-D12d-R (SEQ ID NO: 64 in the Sequence Listing), using an LA Taq HS polymerase Kit (Takara Bio) (PCR cycles: 98° C. 2 min/98° C. 20 sec, 60° C. 30 sec, 72° C. 1.5 min, 30 cycles/4° C.).

Introducing the Δ12 desaturase gene/neomycin-resistant gene expression cassette into T. aureum successfully produced transfectants. These transfectants were subjected to PCR using the genomic DNA as a template, and the result confirmed that the Δ12 desaturase gene and the neomycin-resistant gene were successfully introduced into the genomic DNA of the Δ12 desaturase gene/neomycin-resistant gene expression cassette transfectants (FIG. 34).

vii. Expression of Δ12 Desaturase mRNA in mh0186 Strain

Total RNA was extracted from a main cell culture incubated in a 100-ml GY liquid medium (containing 0.5 mg/ml G418) for 3 days, using a Sepasol RNAISuper (nacalai tesque). The purity of the total RNA was increased by purification, and cDNA was produced as in Example 1, (1)-2. By using the cDNA as a template, a PCR was performed with primers 3F (SEQ ID NO: 52 in the Sequence Listing), 4R (SEQ ID NO: 53 in the Sequence Listing), ubpro-D12d-F (SEQ ID NO: 63 in the Sequence Listing), and ub term-D12d-R (SEQ ID NO: 64 in the Sequence Listing), using an LA Taq HS polymerase kit (Takara Bio) (PCR cycles: 98° C. 2 min/98° C. 20 sec, 60° C. 30 sec, 72° C. 1.5 min, 30 cycles/4° C.).

The result of the PCR using the cDNA as a template indicated that the incorporated Δ12 desaturase gene and neomycin-resistant gene were transcribed into mRNA (FIG. 35).

viii. Expression of Δ12 Desaturase mRNA in T. aureum

Total RNA was extracted from a main cell culture incubated in a 100-ml GY liquid medium (containing 2 mg/ml G418) for 7 days, using a Sepasol RNAISuper (nacalai tesque). The purity of the total RNA was increased by purification, and cDNA was produced as in Example 1, (1)-2. By using the cDNA as a template, a PCR was performed with primers 3F (SEQ ID NO: 52 in the Sequence Listing), 4R (SEQ ID NO: 53 in the Sequence Listing), ubpro-D12d-F (SEQ ID NO: 63 in the Sequence Listing), and ub term-D12d-R (SEQ ID NO: 64 in the Sequence Listing), using an LA Taq HS polymerase kit (Takara Bio) (PCR cycles: 98° C. 2 min/98° C. 20 sec, 60° C. 30 sec, 72° C. 1.5 min, 30 cycles/4° C.).

The result of the PCR using the cDNA as a template indicated that the incorporated Δ12 desaturase gene and neomycin-resistant gene were transcribed into mRNA (FIG. 34).

(3) Expression of Thraustochytrium aureum-Derived Δ5 Desaturase i. Cloning of Δ5 Desaturase

Primers 3F (SEQ ID NO: 65 in the Sequence Listing) and 1R (SEQ ID NO: 66 in the Sequence Listing) were produced in the conserved region present in the sequence of the Δ5 desaturase of Thraustochytrium sp. ATCC 26185, a closely related species of Thraustochytrium aureum ATCC 34304. This was followed by a nested PCR with an Advantage 2 PCR Kit (Clontech), using a T. aureum-derived RACE cDNA library as a template (PCR cycles: 94° C. 30 sec, 50° C. 30 sec, 72° C. 2 min, 30 cycle). As a result, an amplification product of the target size was obtained with a bracketing primer set (1R NES: SEQ ID NO: 67 in the Sequence Listing).

Analysis of the DNA fragment of the expected size (550 bp) obtained with the bracketing primers revealed that the DNA fragment was of the T. aureum Δ5 desaturase. Accordingly, primers with a 100% match (RACEd5F: SEQ ID NO: 68 in the Sequence Listing, and RACEd5FNES: SEQ ID NO: 69 in the Sequence Listing) were produced from the amplified fragment, and RACE PCR was performed using an Advantage 2 PCR Kit (PCR cycles: 94° C. 30 sec, 50° C. 30 sec, 72° C. 2 min, 30 cycle). As a result, a 700-bp 3′-end of the Δ5 desaturase was obtained.

A reverse primer GSP1 (SEQ ID NO: 70 in the Sequence Listing) was produced from this known sequence, and a 5′ RACE PCR was performed (PCR cycles: 94° C. 30 sec/72° C. 3 min, 5 cycles, 94° C. 30 sec/70° C. 30 sec/72° C. 3 min, 5 cycles, 94° C. 30 sec/68° C. 30 sec/72° C. 3 min, 20 cycles). The resulting 5′ RACE product was shorter than the expected size. Thus, instead of the PCR using the cDNA as a template, a PCR using a genome cassette library (TaKaRa LA PCR in vitro Cloning Kit) as a template was performed as above (primer GSP2; SEQ ID NO: 71 in the Sequence Listing). PCR using a BglII cassette library as a template produced a genome sequence about 2.5 kbp upstream of the primer producing site.

The upstream sequence obtained by using the genome walking technique included the start codon for Δ5 desaturase, and there was no presence of introns in the genome analyzed. From the sequences obtained by 3′-RACE or 5′-RACE, the full-length sequence information of Δ5 desaturase was acquired. The full-length sequence consisted of 439 amino acids with a 1,320-bp ORF, and contained a single cytochrome b5 domain (HPGGSI) and three histidine boxes (HECGH, HSKHH, and QIEHH), highly conserved regions of Δ5 desaturase. Based on this information, a PCR was performed with the primers d5fulllengthF (SEQ ID NO: 72 in the Sequence Listing) and d5fulllengthR (SEQ ID NO: 73 in the Sequence Listing) produced at the ORF ends, using the cDNA as a template (PCR cycles: 94° C. for 30 s, 60° C. for 30 s, and 72° C. for 2 min, 30 cycles). As a result, a full-length T. aureum-derived Δ5 desaturase was isolated.

ii. Alignment with Δ5 Desaturases Derived from Other Organisms

Multiple alignment was performed with ClustalX-1.83.1, using the amino acid sequence of T. aureum-derived Δ5 desaturase, and the amino acid sequences of Δ5 desaturases derived from Thraustochytrium sp. ATCC 26185, Dictyostelium discoideum, Rattus norvegicus, Mus musculus, Homo sapiens, Caenorhabditis elegans, and Leishmania major (FIG. 35).

The result showed that the T. aureum-derived Δ5 desaturase at the amino acid level had significant homology with the Δ5 desaturase genes derived from other organisms (D. discoideum: 34%, R. norvegicus: 28%, M. musculus: 28%, H. sapiens: 26%). The homology was particularly high (57%) with the Thraustochytrium sp. belonging to the same genus.

iii. Phylogenetic Analysis

A molecular phylogenetic tree of all desaturase genes, including the T. aureum-derived Δ5 desaturase, was created by using the maximum-likelihood method with molphy. First, the all sequences were prepared into Fasta format, and multiple alignment was performed using clustalW. After removing the uncertain portions, a search was made for a maximum-likelihood phylogenetic tree, using the phylogenetic tree by the neighbor-joining method as the initial phylogenetic tree.

It was found as a result that the acquired gene was close to the protozoa-derived desaturase group, and classified into the same lineage group to which the Δ5 desaturases derived from Thraustochytrium sp. ATCC 26185 and L. major belong (FIG. 36).

iv. Expression of Δ5 Desaturase in Yeast

In order to verify that the acquired gene was Δ5 desaturase, overexpression experiment was conducted using a budding yeast S. cerevisiae as a host. First, the acquired gene was incorporated at the EcoRI/XhoI site of a yeast vector pYES2/CT (Invitrogen) to construct an expression vector pYΔ5des. The constructed expression vector pY5Δdes was then introduced into S. cerevisiae, and GC analysis was performed after extracting and methylating the transfectant fatty acids obtained by using the lithium acetate technique. A gas chromatograph GC-2014 (Shimadzu Corporation) was used for the GC analysis, which was performed under the following conditions. Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.), column temperature: 150° C.→(5° C./min)→220° C. (10 min), carrier gas: He (1.3 mL/min). GC-17A and GCMS-QP-5000 (Shimadzu Corporation) were used for GC-MS analysis, which was performed using a DB-1 capillary column (0.25 mmi.d.×30 m, film thickness 0.25 μm; Agilent) under the following conditions: column temperature 160° C.→(4° C./min)→260° C., injector port temperature 250° C. For peaks that caused troubles in the analyses, the fatty acids were analyzed after picolinyl esterification, using the same apparatuses and columns under the temperature condition 240° C.→(2.5° C./min)→260° C. (15 min)→(2.5° C./min)→280° C.

As a result, eicosatetraenoic acid (ETA; C20:4 Δ8, 11, 14, 17), and dihomo-γ-linoleic acid (DGLA; C20:3 Δ8, 11, 14)—known precursor substances of Δ5 desaturase—were converted into EPA and arachidonic acid (AA; C20:4 n-6), respectively. Conversion efficiencies were 32% and 27%, respectively. No specificity for other substrates was observed. GC-MS confirmed a match in the structures of the conversion products EPA and AA (FIG. 37 a to c, Table 7)

TABLE 7 Percentage of substrate Fatty acid substrates converted (%) C18:3n − 3 (-Linolenic acid (ALA) 0.0 C18:2n − 6 Linoleic acid (LA) 0.0 C20:4n − 3 Eicosatetraenoic acid (ETA) 32.0 C20:3n − 6 Dihomo-g-linolenic acid (DGLA) 27.0 C20:3n − 3 Eicosatrienoic acid 0.0 C20:2n − 6 Eicosadienoic acid 0.0 C22:5n − 3 Docosapentaenoic acid (DPA) 0.0 C22:4n − 6 Docosatetraenoic acid (DTA) 0.0 Conversion rate = (product × 100)/(substrate + product) v. Incorporation of Δ5 Desaturase Gene into mh0186 Genomic DNA

T. aureum ATCC 34304-derived ubiquitin gene promoter/terminator were isolated to construct an expression vector. To begin with, the ubiquitin gene was isolated by using the RACE method, as follows. First, a 3′ fragment of the ubiquitin gene was amplified by PCR with a degenerate primer 2F (SEQ ID NO: 74 in the Sequence Listing), using the cDNA as a template.

Next, a 5′ RACE System for Rapid Amplification of cDNA Ends, version 2.0 (invitrogen) was used to produce a reverse-transcription primer 1R (SEQ ID NO: 17 in the Sequence Listing) and a 5′ RACE primer (SEQ ID NO: 75 in the Sequence Listing), and the kit was operated to obtain a 5′ RACE product.

Based on the ORF sequence of the ubiquitin gene, primers REVERS-U PR-1 (SEQ ID NO: 22 in the Sequence Listing) and REVERS-U PR-2 (SEQ ID NO: 23 in the Sequence Listing) were produced, and a PCR was performed by using the genome walking technique (PCR cycles: 98° C. 30 sec/60° C. 30 sec/72° C. 2 min, 30 cycles). As a result, a 812-bp promoter region was isolated by PCR using a SalI cassette library as a template.

Next, the terminator was isolated using the same method, and a 612-bp DNA fragment was obtained.

Note that the PCR used the primer ubqterminalf1 (SEQ ID NO: 24 in the Sequence Listing) in the 1st PCR, and the primer ter2F (SEQ ID NO: 25 in the Sequence Listing) in the 2nd PCR, and was performed in a PCR cycle consisting of 94° C. 30 sec/60° C. 30 sec/72° C. 3 min, 30 cycles. The amplified fragments were joined by fusion PCR, and incorporated in pUC18 to produce a cyclic vector as shown in FIG. 38 a. The introduced gene fragments shown in FIG. 38 b were then prepared by PCR.

Next, a gene introduction experiment was conducted using Aurantiochytrium sp. mh0186. First, single colonies of the Aurantiochytrium sp. mh0186 strain were cultured in GY medium at 25° C. until the logarithmic growth phase, and the supernatant was removed by centrifugation at 3,500×g, 4° C. for 15 min. Cells (5×10⁶) were suspended in a Nucleofector kit L (amaxa), and pulsated twice with the introduced DNA under 0.75 kV, 50 Ω, 50 μF conditions using a Bio Rad Gene Pulser II (Bio-Rad Laboratories). After quickly adding PD liquid medium (1 ml), a shake culture was performed overnight at 25° C. The cells were then inoculated in a PDA agar plate medium containing 0.5 mg/ml G418, and cultured 3 to 4 days to obtain transfectants.

Then, in order to confirm incorporation of the introduced gene in the genomic DNA of transfectant, a PCR was performed using genomic DNA as a template, using Δ5 desaturase amplification primers d5fulllengthF (SEQ ID NO: 72 in the Sequence Listing) d5fulllengthR (SEQ ID NO: 73 in the Sequence Listing), and neomycin-resistant gene amplification primers FU2FA (SEQ ID NO: 76 in the Sequence Listing) and FU2RA (SEQ ID NO: 77 in the Sequence Listing) (PCR program: 98° C. 10 sec/98° C. 10 sec/60° C. 30 sec/72° C. 1.5 min, 30 cycles).

The result confirmed amplification of the introduced gene, and incorporation in the genome (FIG. 39).

vi. Expression of Δ45 Desaturase mRNA

The Aurantiochytrium sp. mh0186 transfectants were cultured, and RNA extraction was performed according to the protocol attached to the kit (Sepasol RNA I super; nacalai tesque). First, the total RNA obtained from each clone was reverse transcribed to synthesize cDNA, using a PrimeScript Reverse Transcriptase (TakaraBio). Then, a PCR was performed under the following conditions, using the cDNA as a template (PCR cycles: 98° C. 10 sec/55° C. 30 sec/72° C. 1.5 min, 30 cycles).

As a result, amplification of each target gene was confirmed (FIG. 40). The result thus confirmed expression of the introduced gene in the transfectants through transcription into mRNA.

Example 7 Modification of Aurantiochytrium limacinum mh0186 Fatty Acid Composition by Transformation (1) Modification of Fatty Acid Composition by Expression of Pinguiochrysis-Derived Δ12 Desaturase

The transformed clone obtained in Example 6, (2), v. was cultured for 2 days in a 10-ml GY liquid medium (containing 0.5 mg/ml G418), and for an additional day after adding oleic acid to make the final concentration 50 μM. After culturing, the fatty acid composition was analyzed by GC and GC-MS analyses as in Example 6, (2), iv. A gas chromatograph GC-2014 (Shimadzu Corporation) was used for the GC analysis, which was performed under the following conditions. Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.), column temperature: 150° C.→(5° C./min)→220° C. (10 min), carrier gas: He (1.3 mL/min). GC-17A and GCMS-QP-5000 (Shimadzu Corporation) were used for the GC-MS analysis, which was performed using a DB-1 capillary column (0.25 mmi.d.×30 m, film thickness 0.25 μm; Agilent) under the following conditions: column temperature 160° C.→(4° C./min)→260° C., injector port temperature 250° C. For peaks that caused troubles in the analyses, the fatty acids were analyzed after picolinyl esterification, using the same apparatuses and columns under the temperature condition 240° C.→(2.5° C./min)→260° C. (15 min)→(2.5° C./min)→280° C. A transfectant produced by introducing only the neomycin-resistant gene cassette was used as a control.

The GC analysis of the fatty acid compositions of the transfectants confirmed a new peak in the Δ12 desaturase gene/neomycin-resistant gene expression cassette-introduced strain, but not in the control strain, at a position corresponding to the retention time of linoleic acid (FIG. 41). The GC-MS analysis of the new peak revealed that the molecular weight and fragment pattern coincide with those of the sample linoleic acid methyl ester (FIG. 42). The conversion efficiency from oleic acid to linoleic acid was 30.1±6.64%, and there was no effect on other fatty acid compositions (FIG. 43).

(2) Changes in Fatty Acids by Expression of Thraustochytrium aureum-Derived Δ5 Desaturase

The transformed clone obtained in Example 6, (3), v. was cultured for 3 days, and mhneo^(r) and mh45neo^(r) were analyzed by GC analysis after extracting the fatty acid methyl ester. Separately, 0.1 mM ETA or DGLA, exogenous fatty acids used as a substrate by Δ5 desaturase, was added to medium for incorporation into Labyrinthula, and GC analysis was performed after extracting the fatty acids as in the overexpression experiment using yeast. A gas chromatograph GC-2014 (Shimadzu Corporation) was used for the GC analysis, which was performed under the following conditions. Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.), column temperature: 150° C.→(5° C./min)→220° C. (10 min), carrier gas: He (1.3 mL/min).

It was found that the endogenous ETA in the Aurantiochytrium sp. mh0186 strain was converted by the action of the introduced Δ5 desaturase, and that the EPA content was higher than in mhneo^(r) by a factor of about 1.4 (FIG. 44, Table 8). ETA or DGLA used as a substrate by Δ5 desaturase was also added to medium at 0.1 mM for incorporation into Labyrinthula. As a result, the precursor substances converted into EPA and AA in Labyrinthula, and the content increased, as observed in the Δ5 desaturase expression experiment using yeast (Tables 9 and 10). The conversion efficiencies of the precursor substances in Labyrinthula were higher than in yeast, 75.2% and 62.9% for ETA and DGLA, respectively. This experiment was repeated in three or more confirmatory experiments to confirm reproducibility. All experiments produced the same results.

TABLE 8 mhneor (%) mhΔ5neor (%) C14:0 2.23 ± 0.05 2.32 ± 0.03 C15:0 2.43 ± 0.62 2.97 ± 0.96 C16:0 55.2 ± 1.83 52.1 ± 3.15 C17:0 0.97 ± 0.22 1.19 ± 0.42 C18:0 1.54 ± 0.03 1.39 ± 0.13 DGLA ND ND AA 0.18 ± 0.04 0.21 ± 0.02 ETA 0.32 ± 0.02 0.04 ± 0.04 EPA 0.65 ± 0.04 0.94 ± 0.13 DPA 5.17 ± 0.05 5.61 ± 1   DHA 31.3 ± 0.93 33.2 ± 2.44

TABLE 9 mhneor + DGLA (%) mhΔ5neor + DGLA (%) C14:0 2.22 ± 0.06 2.28 ± 0.16 C15:0 2.53 ± 0.63 2.96 ± 0.79 C16:0 53.5 ± 2.36  52 ± 3.41 C17:0 0.99 ± 0.21 1.19 ± 0.41 C18:0 1.56 ± 0.03 1.42 ± 0.13 DGLA* 3.92 ± 0.21 1.09 ± 0.7  AA 0.14 ± 0.01 1.85 ± 0.24 ETA 0.39 ± 0.04 0.08 ± 0.05 EPA  0.6 ± 0.04 1.15 ± 0.29 DPA 4.92 ± 0.11 5.44 ± 0.89 DHA 29.3 ± 1.32 30.5 ± 1.94

TABLE 10 mhneor + ETA (%) mhΔ5neor + ETA (%) C14:0 2.26 ± 0.1  2.43 ± 0.07 C15:0 2.48 ± 0.64 3.04 ± 0.91 C16:0 54.6 ± 1.56 51.8 ± 3.56 C17:0 0.96 ± 0.23 1.17 ± 0.41 C18:0 1.56 ± 0.02  1.4 ± 0.13 DGLA ND ND AA 0.15 ± 0.02 0.22 ± 0.02 ETA* 3.27 ± 0.44 0.94 ± 0.5  EPA 0.62 ± 0.03 2.85 ± 0.35 DPA 4.92 ± 0.06 5.35 ± 0.97 DHA 29.2 ± 0.53 30.8 ± 2.52

Example 8 Labyrinthula Gene Introduction Experiment 2

Gene introduction experiments were conducted using Schizochytrium aggregatum ATCC 28209, Ulkenia sp. ATCC 28207, Schizochytrium sp. SEK210 (NBRC 102615), Schizochytrium sp. SEK345 (NBRC 102616), Botryochytrium radiatum SEK353 (NBRC 104107), and Parietichytrium sarkarianum SEK364.

(1) Determination of MIC in Agar Plate Culture

Precultures (5 μl) of the six Labyrinthulomycetes strains were dropped onto PDA agar plate media containing various concentrations of G418. After culturing the cells at 28° C. for 7 days, colony formation was observed. In consideration of the results of the antibiotic sensitivity test and the selection marker genes used for the eukaryotes transformation system, G418 was found to be effective for the selection marker genes usable in the Labyrinthulomycetes transformation system.

(2) Isolation of T. aureum-Derived Ubiquitin Gene and Gene Expression Regulatory Region

Isolation of the T. aureum-derived ubiquitin gene and the gene expression regulatory region was performed in the same manner as in Example 3.

(3) Production of Drug-Resistant Gene Expression Cassette

The drug-resistant gene expression cassette was produced in the same manner as in Example 4.

(4) Gene Introduction Experiment

A gene introduction experiment was conducted using a linear Neo^(r) expression cassette (ub-Neo^(r)) adopting the ubiquitin promoter and terminator. The cassette was produced by performing a PCR with an oligonucleotide primer set NeoF (SEQ ID NO: 78 in the Sequence Listing)/NeoR (SEQ ID NO: 79 in the Sequence Listing), using an LA taq Hot Start Version (Takara Bio), and the pUBNeomycin r obtained in Example 4-ii. as a template, and the resulting amplification product was gel purified.

The gene introduction experiment was performed by electroporation. Specifically, Labyrinthulomycetes were cultured in a GY liquid medium or H liquid medium to the early to late stage of the logarithmic growth phase at 28° C., 150 rpm, and the supernatant was removed by centrifugation at 3,500×g, 4° C. for 10 min. The resulting cells were suspended in sterilized 1.75% Sea Life (Marine Tech), and washed by recentrifugation. The cells (5×10⁶) were then suspended with the introduced DNA ub-Neo^(r) in a reagent NucleofectorR solution L for gene introduction (amaxa). This was followed by application of electrical pulses using a Gene Pulser (Bio-Rad Laboratories; 1-mm gap cuvette; pulse settings: 50 μF/50 Ω/0.75 kV, applied twice). After applying electrical pulses, GY liquid medium (1 ml) was immediately added, and the cells were cultured at 28° C. for 12 hours. The culture fluid was then applied to a PDA agar plate medium containing 2.0 mg/ml G418 (Ulkenia sp. ATCC 28207, Schizochytrium sp. SEK210, and Parietichytrium sarkarianum SEK364), or 1.0 mg/ml G418 (Botryochytrium radiatum SEK353, Schizochytrium aggregatum ATCC 28209, and Schizochytrium sp. SEK345). After static culturing at 28° C., colony formation of transfectants with the conferred G418 resistance was observed.

As a result, colonies with the conferred G418 resistance were observed for the linear DNA ub-Neo^(r) at the efficiency as high as 1.6×10⁰ cfu/μg DNA. It was found that the transfectants maintained the G418 resistance even after being subcultured five times in a GY liquid medium containing no G418. The result using G418 resistance as an index thus confirmed that the conferred character was stable.

(5) Evaluation of Transfectant by PCR Using Genomic DNA as Template

The transfectants were cultured in GY liquid media containing 1.0 and 2.0 mg/ml G418. The wild-type strain was cultured in a GY liquid medium containing no G418. Genomic DNA was extracted from the cells of these strains using an ISOPLANT (nacalai tesque). Neo^(r) was then amplified by PCR using a KOD FX (Toyobo life science), using the genomic DNA as a template. Oligonucleotide primers NeoF (SEQ ID NO: 78 in the Sequence Listing)/NeoR (SEQ ID NO: 79 in the Sequence Listing) were used (PCR cycles: 94° C. 2 min/98° C. 10 sec, 68° C. 30 sec, 72° C. 2 min, 30 cycles/4° C.). As a result, specific Neo^(r) amplification, not found in the wild-type strain, was observed in the transfectants (FIG. 45). The result thus suggested that the introduced ub-Neo^(r) was incorporated in the chromosomal DNA.

Example 9 Expression of ω3 Desaturase Gene in Thraustochytrium aureum Example 9-1 Subcloning of SV40 Terminator Sequence

An SV40 terminator sequence was amplified with PrimeSTAR polymerase (Takara Bio), using a pcDNA 3.1 Myc-His vector as a template. The following PCR primers were used. RHO58 was set on the SV40 terminator sequence, and included BglII and BamHI linker sequences. RHO52 was set on the SV40 terminator sequence, and included a BglII sequence. [RHO58: 34 mer: 5′-CAG ATC TGG ATC CGC GAA ATG ACC GAC CAA GCG A-3′ (SEQ ID NO: 80), RHO52: 24 mer: 5′-ACG CAA TTA ATG TGA GAT CTA GCT-3′ (SEQ ID NO: 81)]. After amplification performed under the conditions below, the product was cloned into a pGEM-T easy vector (Promega). [PCR cycles: 98° C. 2 min/98° C. 30 sec, 60° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 1 min]. After amplification with Escherichia coli, the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit. This was named pRH27.

The plasmid (pRH27) containing the subcloned SV40 terminator sequence (342 bp, SEQ ID NO: 82) is shown in FIG. 46.

Example 9-2 Production of Blasticidin Resistant Gene Cassette

A ubiquitin promoter sequence (618 bp, SEQ ID NO: 83) was amplified from Thraustochytrium aureum ATCC 34304 with a PrimeSTAR GC polymerase, using genomic DNA as a template. The following PCR primers were used. RHO53 was set on the ubiquitin promoter sequence, and included a BglII linker sequence. RHO48 included a ubiquitin promoter sequence and a blasticidin resistant gene sequence. [RHO53: 36 mer: 5′-CCC AGA TCT GCC GCA GCG CCT GGT GCA CCC GCC GGG-3′ (SEQ ID NO: 84), RHO48: 58 mer: 5′-CTT CTT GAG ACA AAG GCT TGG CCA TGT TGG CTA GTG TTG CTT AGG TCG CTT GCT GCT G-3′ (SEQ ID NO: 85)]. [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30 cycles/68° C. 1 min].

The blasticidin resistant gene (432 bp, SEQ ID NO: 86) was amplified with a PrimeSTAR GC polymerase, using pTracer-CMV/Bsd/lacZ as a template. The following PCR primers were used. RHO47 included a ubiquitin promoter sequence and a blasticidin resistant gene sequence. RHO49 included a blasticidin resistant gene sequence, and had a BglII linker sequence. [RHO47: 54 mer: 5′-AGC GAC CTA AGC AAC ACT AGC CAA CAT GGC CAA GCC TTT GTC TCA AGA AGA ATC-3′ (SEQ ID NO: 87), RHO49: 38 mer: 5′-CCC AGA TCT TAG CCC TCC CAC ACA TAA CCA GAG GGC AG-3′ (SEQ ID NO: 88)]. [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30 cycles/68° C. 1 min].

Fusion PCR was performed with RHO53 (SEQ ID NO: 85) and RHO49 (SEQ ID NO: 88), using SEQ ID NOS: 83 and 86 as templates. LA taq Hot start version was used as the enzyme, and the amplification was performed under the following conditions. [PCR cycles: 94° C. 2 min/94° C. 20 sec, 55° C. 30 sec, 68° C. 1 min, 30 cycles/68° C. 1 min; 1° C./10 sec from 55° C. to 68° C.] (FIG. 47).

The Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter-pTracer-CMV/Bsd/lacZ-derived blasticidin resistant gene (1,000 bp, SEQ ID NO: 89) fused as above was digested with BglII, and ligated at the BamHI site of pRH27 (FIG. 46) described in Example 9-1. The resulting plasmid was amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit. This was named pRH38.

The product blasticidin resistant gene cassette (pRH38) is shown in FIG. 48.

Example 9-3 Cloning of Saprolegnia diclina-Derived ω3 Desaturase Gene, and Production of Gene Expression Plasmid

A ubiquitin promoter sequence (longer) (812 bp, SEQ ID NO: 90) was amplified from with an LA taq GC II polymerase, using genomic DNA of Thraustochytrium aureum ATCC 34304 as a template. The following PCR primers were used. TMO42 was set on the ubiquitin promoter sequence, upstream of RE1053 (Example 9-2, SEQ ID NO: 84), and included a KpnI linker sequence. TMO43 included a ubiquitin promoter sequence and a Saprolegnia diclina-derived ω3 desaturase gene sequence. [TMO42: 29 mer: 5′-TCG GTA CCC GTT AGA ACG CGT AAT ACG AC-3′ (SEQ ID NO: 91), TMO43: 45 mer: 5′-TTC GTC TTA TCC TCA GTC ATG TTG GCT AGT GTT GCT TAG GTC GCT-3′ (SEQ ID NO: 92)]. [PCR cycles: 96° C. 2 min/98° C. 20 sec, 60° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 1 min].

The Saprolegnia diclina was then cultured in a medium containing D-glucose (31.8 g) and a yeast extract (10.6 g) per liter (adjusted with deionized water). Cells in the late stage of the logarithmic growth phase were centrifuged at 4° C., 3,500×g for 5 min to prepare pellets, and freeze disrupted with liquid nitrogen. The disrupted cell solution was extracted with phenol. After ethanol precipitation, the precipitate was dissolved in TE solution. The nucleic acids dissolved in the TE solution were treated with RNase at 37° C. for 30 min to degrade RNA, and extracted again with phenol. After ethanol precipitation, the precipitate was dissolved in TE solution. The DNA purity and concentration were calculated by measuring A260/280. The Saprolegnia diclina-derived ω3 desaturase gene sequence (1,116 bp, SEQ ID NO: 93) was amplified with an LA taq GC II polymerase, using the genomic DNA of the Saprolegnia diclina as a template. The following PCR primers were used. TMO44 included a ubiquitin promoter sequence and a Saprolegnia diclina-derived ω3 desaturase gene sequence. TMO45 included a Saprolegnia diclina-derived ω3 desaturase gene sequence and a ubiquitin terminator. [TMO44: 43 mer: 5′-CCT AAG CAA CAC TAG CCA ACA TGA CTG AGG ATA AGA CGA AGG T-3′ (SEQ ID NO: 94), TMO45: 40 mer: 5′-ATA CTA CAG ATA GCT TAG TTT TAG TCC GAC TTG GCC TTG G-3′ (SEQ ID NO: 95)]. [PCR cycles: 96° C. 2 min/98° C. 20 sec, 60° C. 30 sec, 72° C. 1 min 30 sec, 30 cycles/72° C. 1 min 30 sec].

The ubiquitin terminator sequence (614 bp, SEQ ID NO: 96) was amplified with an LA taq GC II polymerase, using the genomic DNA of Thraustochytrium aureum ATCC 34304 as a template. The following PCR primers were used. TMO46 included a Saprolegnia diclina-derived ω3 desaturase gene sequence and a ubiquitin terminator. TMO47 was designed on the ubiquitin terminator sequence, and included a KpnI linker sequence. [TMO46: 44 mer: 5′-CCA AGG CCA AGT CGG ACT AAA ACT AAG CTA TCT GTA GTA TGT GC-3′ (SEQ ID NO: 97), TMO47: 45 mer: 5′-TCG GTA CCA CCG CGT AAT ACG ACT CAC TAT AGG GAG ACT GCA GTT-3′ (SEQ ID NO: 98)]. [PCR cycles: 96° C. 2 min/98° C. 20 sec, 60° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 1 min].

Fusion PCR was performed with TMO42 (SEQ ID NO: 91) and TMO47 (SEQ ID NO: 98), using SEQ ID NOS: 90, 93, and 96 as templates. LA taq GC II polymerase was used as the enzyme, and the amplification was performed under the following conditions. [PCR cycles: 96° C. 2 min/98° C. 20 sec, 55° C. 30 sec, 68° C. 3 min, 30 cycles/68° C. 3 min; 1° C./10 sec from 55° C. to 68° C.] (FIG. 49, 2,463 bp, SEQ ID NO: 99).

A PCR was performed with RHO84 (SEQ ID NO: 100, presented below) and RHO52 (Example 9-1, SEQ ID NO: 101), using the pRH38 (FIG. 48) described in Example 9-2 as a template. RHO84 was set on the ubiquitin promoter, and had a KpnI linker sequence. RHO52 was set on the SV40 terminator sequence, and had a BglII linker. LA taq Hot start version was used as the enzyme, and the amplification was performed under the following conditions, and cloned into a pGEM-T easy vector. [RHO84: 36 mer: 5′-CCC GGT ACC GCC GCA GCG CCT GGT GCA CCC GCC GGG-3′ (SEQ ID NO: 100)]. [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 1 min 30 sec, 30 cycles/68° C. 3 min]. After amplification with Escherichia coli, the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit. This was named pRH45 (FIG. 50).

The fused Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter-Saprolegnia diclina-derived ω3 desaturase gene-Thraustochytrium aureum ATCC 34304-derived ubiquitin terminator (SEQ ID NO: 99; FIG. 49) was digested with KpnI, and ligated at the KpnI site of the pRH45 (FIG. 50). The resulting plasmid was amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit. This was named pRH48.

The product Saprolegnia diclina-derived ω3 desaturase gene expression plasmid (pRH48) is shown in FIG. 51.

Example 9-4

Introduction of Saprolegnia diclina-Derived ω3 Desaturase Expression Plasmid into Thraustochytrium aureum DNA was amplified using a Prime STAR Max polymerase with primers TMO42 (Example 9-3, SEQ ID NO: 91) and RHO52 (Example 9-1, SEQ ID NO: 81), using the targeting vector produced in Example 9-3 as a template. [PCR cycles: 94° C. 30 sec, 72° C. 1 min, 5 cycles/94° C. 30 sec, 70° C. 30 sec, 72° C. 1 min, 5 cycles/94° C. 30 sec, 68° C. 30 sec, 72° C. 1 min, 25 cycles/72° C. 2 min]. The amplification product was collected from 1.0% agarose gel, and, after ethanol precipitation, the precipitate was dissolved in 0.1×TE. The DNA concentration was calculated by measuring A260/280. The fragment amplified by PCR was 3,777 bp, and contained the ubiquitin promoter-ω3 desaturase gene-ubiquitin terminator-ubiquitin promoter-blasticidin resistant gene sequence- and SV40 terminator sequence in this order (SEQ ID NO: 101).

Thraustochytrium aureum was cultured in a GY medium for 4 days, and cells in the logarithmic growth phase were used for gene introduction. A DNA fragment (0.625 μg) was introduced into cells corresponding to OD₆₀₀=1 to 1.5, using the gene gun technique (microcarrier: 0.6-micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupture disk: 1,100 PSI). After a 4- to 6-hour recovery time, the gene introduced cells were applied onto a 0.2 mg/ml blasticidin-containing PDA agar plate medium.

Twenty to thirty drug-resistant strains were obtained per penetration.

Example 9-5 Acquisition of Saprolegnia diclina-Derived ω3 Desaturase Gene Expressing Strain

Genomic DNA was extracted from the ω3 desaturase gene expressing strain obtained in Example 9-4, and the DNA concentration was calculated by measuring A260/280. By using this as a template, a PCR was performed to confirm the genome structure, using an LA taq Hot start version. The positions of the primers, combinations used for the amplification, and the expected size of the amplification product are shown in FIG. 52. TMO42 (Example 9-3, SEQ ID NO: 91) was set on the ubiquitin promoter, and RHO49 (Example 9-2, SEQ ID NO: 88) on the blasticidin resistant gene. [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 4 min, 30 cycles/68° C. 7 min].

The result of amplification confirmed a band of an expected size (FIG. 53). That is, a strain was isolated that contained the introduced expression fragment stably introduced into its genome.

Example 9-6 Changes in Fatty Acid Composition by Expression of ω3 Desaturase in Thraustochytrium aureum

The Thraustochytrium aureum, and the ω3 desaturase expressing strain obtained in Example 9-5 were cultured. After freeze drying, the fatty acids were subjected to methylesterification, and analyzed by GC analysis. A gas chromatograph GC-2014 (Shimadzu Corporation) was used for the GC analysis, which was performed under the following conditions. Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.), column temperature: 150° C.-4 (5° C./min)-220° C. (10 min), carrier gas: He (1.3 mL/min).

The ω3 desaturase expressing strain had reduced levels of the n-6 series fatty acids, and there was a tendency for the n-3 series fatty acids to increase (FIG. 54). FIG. 55 represents the percentage relative to the wild-type strain taken as 100%.

As a result, the arachidonic acid was reduced by about 1/10, and the DPA by about 1/7. EPA increased by a factor of about 1.8, and DHA by a factor of about 1.2.

INDUSTRIAL APPLICABILITY

The present invention provides modification of the fatty acid composition produced by stramenopiles, and a method for highly accumulating fatty acids in stramenopiles. The invention thus enables more efficient production of polyunsaturated fatty acids. 

1. A method for modifying the fatty acid composition of stramenopiles, comprising: introducing a fatty acid desaturase gene into stramenopiles selected from the group consisting of Schizochytrium sp. M-8 strain (FERM P-19755), Thraustochytrium aureum ATCC 34304, Thraustochytrium sp. ATCC 26185, Schizochytrium sp. AL1Ac, Schizochytrium aggregatum ATCC 28209, Ulkenia sp. ATCC 28207, Schizochytrium sp. SEK210 (NBRC 102615), Schizochytrium sp. SEK345 (NBRC 102616), Botryochytrium radiatum SEK353 (NBRC 104107), and Parietichytrium sarkarianum SEK364 (FERM ABP-11298); and expressing the fatty acid desaturase.
 2. The method according to claim 1, wherein the fatty acid desaturase is a desaturase.
 3. The method according to claim 2, wherein the desaturase is a Δ5 desaturase, a Δ12 desaturase, or an ω3 desaturase.
 4. A method for highly accumulating an unsaturated fatty acid in stramenopiles by using the method of any one of claims 1 to
 3. 5. The method according to claim 4, wherein the unsaturated fatty acid is an unsaturated fatty acid of 18 to 22 carbon atoms. 6.-22. (canceled) 